U.S. patent application number 13/837539 was filed with the patent office on 2014-04-17 for drug delivery conjugates containing unnatural amino acids and methods for using.
This patent application is currently assigned to ENDOCYTE, INC.. The applicant listed for this patent is Endocyte, Inc.. Invention is credited to Christopher P. LEAMON, Iontcho R. VLAHOV.
Application Number | 20140107316 13/837539 |
Document ID | / |
Family ID | 50475917 |
Filed Date | 2014-04-17 |
United States Patent
Application |
20140107316 |
Kind Code |
A1 |
VLAHOV; Iontcho R. ; et
al. |
April 17, 2014 |
DRUG DELIVERY CONJUGATES CONTAINING UNNATURAL AMINO ACIDS AND
METHODS FOR USING
Abstract
Described herein are drug delivery conjugates for targeted
therapy. In particular, described herein are drug delivery
conjugates that include polyvalent linkers comprising one or more
unnatural amino acids.
Inventors: |
VLAHOV; Iontcho R.; (West
Lafayette, IN) ; LEAMON; Christopher P.; (West
Lafayette, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Endocyte, Inc.; |
|
|
US |
|
|
Assignee: |
ENDOCYTE, INC.
West Lafayette
IN
|
Family ID: |
50475917 |
Appl. No.: |
13/837539 |
Filed: |
March 15, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61714565 |
Oct 16, 2012 |
|
|
|
61790234 |
Mar 15, 2013 |
|
|
|
Current U.S.
Class: |
530/329 ;
530/330 |
Current CPC
Class: |
A61K 47/64 20170801;
A61K 47/551 20170801; A61K 47/542 20170801 |
Class at
Publication: |
530/329 ;
530/330 |
International
Class: |
A61K 47/48 20060101
A61K047/48 |
Claims
1. A compound of the formula BLD.sub.X, or a pharmaceutically
acceptable salt thereof, wherein B is a cell surface receptor
targeting ligand, D is in each instance an independently selected
drug, x is an integer selected from 1, 2, 3, 4 and 5; and L is a
releasable polyvalent linker comprising one or more unnatural amino
acids; and where B iscovalently attached to L, and L is covalently
attached to each of D; and where that the compound is not of the
formula ##STR00086## or a pharmaceutically acceptable salt
thereof.
2. The compound of claim 1 wherein at least one unnatural amino
acid has the D-configuration.
3. The compound of claim 1 wherein at least one unnatural amino
acid is selected from D-alanine, D-aspartic acid, D-asparagine,
D-cysteine, D-glutamic acid, D-phenylalanine, D-histidine,
D-isoleucine, D-lysine, D-leucine, D-methionine, D-proline,
D-glutamine, D-arginine, D-serine, D-threonine, D-valine,
D-tryptophan, D-tyrosine, and D-ornithine, or a derivative
thereof
4. The compound of claim 1 wherein at least one unnatural amino
acid is selected from D-aspartic acid, D-asparagine, D-cysteine,
D-glutamic acid, D-histidine, D-lysine, D-methionine, D-glutamine,
D-arginine, D-serine, D-threonine, D-tryptophan, D-tyrosine, and
D-ornithine, or a derivative thereof.
5. The compound of claim 1 wherein at least one unnatural amino
acid is selected from D-aspartic acid, D-asparagine, D-cysteine,
D-glutamic acid, D-histidine, D-lysine, D-glutamine, D-arginine,
D-serine, D-threonine, D-tryptophan, and D-ornithine, or a
derivative thereof.
6. The compound of claim 1 wherein at least one unnatural amino
acid is selected from D-aspartic acid, D-cysteine, D-glutamic acid,
D-lysine, D-arginine, D-serine, and D-ornithine, or a derivative
thereof.
7. The compound of claim 1 wherein L comprises two or more
unnatural amino acids.
8. The compound of claim 1 wherein L comprises three or more
unnatural amino acids.
9. The compound of claim 1 wherein L comprises four or more
unnatural amino acids.
10. The compound of claim 1 wherein L further comprises one or more
disulfides.
11. The compound of claim 1 wherein at least one disulfide
comprises D-cysteinyl.
12. The compound of claim 1 wherein L further comprises one or more
divalent hydrophilic radicals.
13. The compound of claim 1 wherein L further comprises two or more
divalent hydrophilic radicals.
14. The compound of claim 1 wherein L further comprises three or
more divalent hydrophilic radicals.
15. The compound of claim 1 wherein L further comprises four or
more divalent hydrophilic radicals.
16. The compound of claim 1 wherein L further comprises one or more
divalent polyoxy radicals.
17. The compound of claim 1 wherein L further comprises two or more
divalent polyoxy radicals.
18. The compound of claim 1 wherein L further comprises three or
more divalent polyoxy radicals.
19. The compound of claim 1 wherein L further comprises four or
more divalent polyoxy radicals.
20. The compound of claim 1 wherein L further comprises one or more
divalent polyhydroxy radicals.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit, under 35 U.S.C.
.sctn.119(e), of U.S. Provisional Application No. 61/714,565, which
was filed Oct. 16, 2012 and U.S. Provisional Application
61/790,234, which was filed Mar. 15, 2013, the entirety of each of
the disclosures of which are hereby incorporated herein by
reference.
TECHNICAL FIELD
[0002] The invention described herein pertains to drug delivery
conjugates for targeted therapy. In particular, the invention
described herein pertains to drug delivery conjugates that include
polyvalent linkers comprising one or more unnatural amino
acids.
BACKGROUND AND SUMMARY OF THE INVENTION
[0003] It has been discovered that drug delivery conjugates that
include polyvalent linkers formed from one or more unnatural amino
acids are efficacious in treating pathogenic cell populations,
including but not limited to cancer, and for treating inflammation,
including but not limited to arthritis.
[0004] In one illustrative embodiment of the invention, compounds
of the formula
B-L-D.sub.X
are described herein.
[0005] In another embodiment, pharmaceutical compositions
containing one or more of the compounds are also described herein.
In one aspect, the compositions include a therapeutically effective
amount of the one or more compounds for treating a patient with
cancer, inflammation, and the like. It is to be understood that the
compositions may include other component and/or ingredients,
including, but not limited to, other therapeutically active
compounds, and/or one or more carriers, diluents, excipients, and
the like. In another embodiment, methods for using the compounds
and pharmaceutical compositions for treating patients with cancer,
inflammation, and the like are also described herein. In one
aspect, the methods include the step of administering one or more
of the compounds and/or compositions described herein to a patient
with cancer, inflammation, and the like. In another aspect, the
methods include administering a therapeutically effective amount of
the one or more compounds and/or compositions described herein for
treating patients with cancer, inflammation, and the like. In
another embodiment, uses of the compounds and compositions in the
manufacture of a medicament for treating patients with cancer,
inflammation, and the like are also described herein. In one
aspect, the medicaments include a therapeutically effective amount
of the one or more compounds and/or compositions for treating a
patient with cancer, inflammation, and the like.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] FIG. 1A shows alone in vivo activity of EC1456 against KB
tumors in nu/nu mice dosed at 1 .mu.mol/kg three times per week
(M/W/F) (TIW) for two consecutive weeks ( ), compared to EC1456
co-dosed with EC0923 at 100 .mu.mol/kg (.tangle-solidup.), and
untreated (PBS) controls (.box-solid.). Dotted vertical line
represents the day of final dose. FIG. 1B shows that EC1456 did not
result in any observable gross toxicity as determined by animal
body weight.
[0007] FIG. 2A shows the activity of EC1456 Against Established
Subcutaneous MDA-MB-231 Tumors. Animals bearing s.c. MDA-MB-231
tumors (94-145 mm.sup.3) were treated i.v. starting on Day 17 with
2 .mu.mmol/kg (panel A) of EC1456 ( ), three times per week (M/W/F)
for a 2 week period compared to untreated animals (.box-solid.), as
shown in FIG. 2A. N=5 animals per cohort. Dotted vertical line=day
of final dose. FIG. 2B shows that EC1456 did not cause gross whole
animal toxicity as determined by % weight change.
[0008] FIG. 3A shows the activity of EC1456 in animals bearing s.c.
KB-CR tumors (98-148 mm3), where EC1456 was administered i.v.
starting on Day 6 with 2 .mu.mol/kg ( ), three times per week
(M/W/F) for a 2 week period, or with 3 mg/kg of cisplatin
(.tangle-solidup.), twice per week (T/Th) for a 2 week period, and
compared to untreated controls (.box-solid.), N=5 animals per
cohort. Dotted vertical line=day of final dosing day. FIG. 3B shows
that EC1456 did not exhibit significant host animal toxicity, and
that cisplatin did exhibit significant host animal toxicity.
[0009] FIG. 4A shows the activity of EC1663 in animals bearing s.c.
KB tumors, where EC1663 was administered i.v. starting on Day 7
with 0.5 .mu.mol/kg (.tangle-solidup.), three times per week
(M/W/F) for a 2 week period, for a 2 week period, and compared to
untreated controls ( ), N=5 animals per cohort. Dotted vertical
line=day of final dosing day. FIG. 4B shows that EC1663 did not
exhibit significant host animal toxicity.
DETAILED DESCRIPTION
[0010] Several illustrative embodiments of the invention are
described by the following clauses:
[0011] A compound of the formula BLD.sub.X, or a pharmaceutically
acceptable salt thereof, wherein B is a cell surface receptor
targeting ligand, D is in each instance an independently selected
drug, x is an integer selected from 1, 2, 3, 4 and 5; and L is a
releasable polyvalent linker comprising one or more unnatural amino
acids; and where B is covalently attached to L, and L is covalently
attached to each of D; and
[0012] where that the compound is not of the formula
##STR00001##
or a pharmaceutically acceptable salt thereof.
[0013] The compound of the preceding clause wherein at least one
unnatural amino acid has the D-configuration.
[0014] The compound of any one of the preceding clauses wherein at
least one unnatural amino acid is selected from D-alanine,
D-aspartic acid, D-asparagine, D-cysteine, D-glutamic acid,
D-phenylalanine, D-histidine, D-isoleucine, D-lysine, D-leucine,
D-methionine, D-proline, D-glutamine, D-arginine, D-serine,
D-threonine, D-valine, D-tryptophan, D-tyrosine, and D-ornithine,
or a derivative thereof.
[0015] The compound of any one of the preceding clauses wherein at
least one unnatural amino acid is selected from D-aspartic acid,
D-asparagine, D-cysteine, D-glutamic acid, D-histidine, D-lysine,
D-methionine, D-glutamine, D-arginine, D-serine, D-threonine,
D-tryptophan, D-tyrosine, and D-ornithine, or a derivative
thereof.
[0016] The compound of any one of the preceding clauses wherein at
least one unnatural amino acid is selected from D-aspartic acid,
D-asparagine, D-cysteine, D-glutamic acid, D-histidine, D-lysine,
D-glutamine, D-arginine, D-serine, D-threonine, D-tryptophan, and
D-ornithine, or a derivative thereof.
[0017] The compound of any one of the preceding clauses wherein at
least one unnatural amino acid is selected from D-aspartic acid,
D-cysteine, D-glutamic acid, D-lysine, D-arginine, D-serine, and
D-ornithine, or a derivative thereof.
[0018] The compound of any one of the preceding clauses wherein L
comprises two or more unnatural amino acids.
[0019] The compound of any one of the preceding clauses wherein L
comprises three or more unnatural amino acids.
[0020] The compound of any one of the preceding clauses wherein L
comprises four or more unnatural amino acids.
[0021] The compound of any one of the preceding clauses wherein L
further comprises one or more disulfides.
[0022] The compound of any one of the preceding clauses wherein at
least one disulfide comprises D-cysteinyl.
[0023] The compound of any one of the preceding clauses wherein L
further comprises one or more divalent hydrophilic radicals.
[0024] The compound of any one of the preceding clauses wherein L
further comprises two or more divalent hydrophilic radicals.
[0025] The compound of any one of the preceding clauses wherein L
further comprises three or more divalent hydrophilic radicals.
[0026] The compound of any one of the preceding clauses wherein L
further comprises four or more divalent hydrophilic radicals.
[0027] The compound of any one of the preceding clauses wherein L
further comprises one or more divalent polyoxy radicals.
[0028] The compound of any one of the preceding clauses wherein L
further comprises two or more divalent polyoxy radicals.
[0029] The compound of any one of the preceding clauses wherein L
further comprises three or more divalent polyoxy radicals.
[0030] The compound of any one of the preceding clauses wherein L
further comprises four or more divalent polyoxy radicals.
[0031] The compound of any one of the preceding clauses wherein L
further comprises one or more divalent polyhydroxy radicals.
[0032] The compound of any one of the preceding clauses wherein L
further comprises two or more divalent polyhydroxy radicals.
[0033] The compound of any one of the preceding clauses wherein L
further comprises three or more divalent polyhydroxy radicals.
[0034] The compound of any one of the preceding clauses wherein L
further comprises four or more divalent polyhydroxy radicals.
[0035] The compound of any one of the preceding clauses wherein at
least one unnatural amino acid comprises a polyhydroxy radical.
[0036] The compound of any one of the preceding clauses wherein at
least two unnatural amino acids comprise a polyhydroxy radical.
[0037] The compound of any one of the preceding clauses wherein at
least three unnatural amino acids comprise a polyhydroxy
radical.
[0038] The compound of any one of the preceding clauses wherein at
least four unnatural amino acids comprise a polyhydroxy
radical.
[0039] The compound of any one of the preceding clauses wherein at
least one of the polyhydroxy radicals is of the formula
CH.sub.2--(CH(OH)).sub.n--CH.sub.2--OH
where n is selected from 1, 2, 3, 4, 5, and 6.
[0040] The compound of any one of the preceding clauses wherein n
is selected from 1, 2, 3, and 4.
[0041] The compound of any one of the preceding clauses wherein n
is selected from 3 and 4.
[0042] The compound of any one of the preceding clauses wherein L
comprises a divalent polyglutamic acid radical, where at least one
glutamic acid forms an amide with an aminopolyhydroxy radical.
[0043] The compound of any one of the preceding clauses wherein L
comprises a divalent polyglutamic acid radical, where at least two
glutamic acids form an amide with an aminopolyhydroxy radical.
[0044] The compound of any one of the preceding clauses wherein L
comprises a divalent polyglutamic acid radical, where at least
three glutamic acids form an amide with an aminopolyhydroxy
radical.
[0045] The compound of any one of the preceding clauses wherein L
comprises a divalent polyglutamic acid radical, where at least four
glutamic acids form an amide with an aminopolyhydroxy radical.
[0046] The compound of any one of the preceding clauses wherein L
comprises a divalent poly(D-glutamic acid) radical, where at least
one glutamic acid forms an amide with an aminopolyhydroxy
radical.
[0047] The compound of any one of the preceding clauses wherein L
comprises a divalent poly(D-glutamic acid) radical, where at least
two glutamic acids form an amide with an aminopolyhydroxy
radical.
[0048] The compound of any one of the preceding clauses wherein L
comprises a divalent poly(D-glutamic acid) radical, where at least
three glutamic acids form an amide with an aminopolyhydroxy
radical.
[0049] The compound of any one of the preceding clauses wherein L
comprises a divalent poly(D-glutamic acid) radical, where at least
four glutamic acids form an amide with an aminopolyhydroxy
radical.
[0050] The compound of any one of the preceding clauses wherein at
least one of the aminopolyhydroxy radicals is of the formula
NH--CH.sub.2--(CH(OH)).sub.m--CH.sub.2--OH
where m is selected from 1, 2, 3, 4, 5, and 6.
[0051] The compound of any one of the preceding clauses wherein L
comprises a divalent radical of the formula
S--CH.sub.2CH.sub.2--O--C(O).
[0052] The compound of any one of the preceding clauses wherein L
comprises a divalent radical of the formula
S--S--CH.sub.2CH.sub.2--O--C(O).
[0053] The compound of any one of the preceding clauses wherein m
is selected from 1, 2, 3, and 4.
[0054] The compound of any one of the preceding clauses wherein m
is selected from 3 and 4.
[0055] The compound of any one of the preceding clauses wherein B
is a folate receptor binding moiety.
[0056] The compound of any one of the preceding clauses wherein B
is a folate.
[0057] The compound of any one of the preceding clauses wherein B
is a folate comprising D-glutamyl.
[0058] The compound of any one of the preceding clauses wherein B
is folate.
[0059] The compound of any one of the preceding clauses wherein B
is an unnatural folate radical of the formula
##STR00002##
[0060] The compound of any one of the preceding clauses wherein x
is 3.
[0061] The compound of any one of the preceding clauses wherein x
is 2.
[0062] The compound of any one of the preceding clauses wherein x
is 1.
[0063] The compound of any one of the preceding clauses wherein at
least one D is a cytotoxic agent.
[0064] The compound of any one of the preceding clauses wherein at
least one D is a cancer treating agent.
[0065] The compound of any one of the preceding clauses wherein at
least one D is an anti-inflammatory agent.
[0066] The compound of any one of the preceding clauses wherein at
least one D is a vinca alkaloid.
[0067] The compound of any one of the preceding clauses wherein at
least one D is desacetylvinblastine monohydrazide.
[0068] The compound of any one of the preceding clauses wherein at
least one D is a tubulysin.
[0069] The compound of any one of the preceding clauses wherein at
least one D is tubulysin A.
[0070] The compound of any one of the preceding clauses wherein at
least one D is tubulysin B.
[0071] The compound of any one of the preceding clauses wherein at
least one D is tubulysin A hydrazide.
[0072] The compound of any one of the preceding clauses wherein at
least one D is tubulysin B hydrazide.
[0073] The compound of any one of the preceding clauses wherein at
least one D is an aminopterin.
[0074] The compound of any one of the preceding clauses wherein B-L
is a radical of the formula
##STR00003##
[0075] The compound of any one of the preceding clauses wherein B-L
is a radical of the formula
##STR00004##
[0076] The compound of any one of the preceding clauses wherein at
least one D is a radical of the formula
##STR00005##
[0077] The compound of any one of the preceding clauses wherein at
least one D is a radical of the formula
##STR00006##
[0078] The compound of any one of the preceding clauses wherein at
least one D is a radical of the formula
##STR00007##
where n=1, 2, 3, 4, 5, or 6, or alternatively, n=1, 2, or 3, or
alternatively, n=2 or 3.
[0079] The compound of any one of the preceding clauses wherein at
least one D is a radical of the formula
##STR00008##
where n=1, 2, 3, 4, 5, or 6, or alternatively, n=1, 2, or 3, or
alternatively, n=2 or 3.
[0080] The compound of any one of the preceding clauses wherein at
least one D is a radical of the formula
[0081] The compound of any one of the preceding clauses wherein at
least one D is a radical of the formula
[0082] The compound of any one of the preceding clauses wherein at
least one D is a radical of the formula
[0083] The compound of any one of the preceding clauses wherein at
least one D is a radical of the formula
[0084] The compound of any one of the preceding clauses wherein the
compound is of the formula EC1456
##STR00009##
or a pharmaceutically acceptable salt thereof.
[0085] The compound of any one of the preceding clauses wherein the
compound is not of the formula
##STR00010##
or a pharmaceutically acceptable salt thereof.
[0086] The compound of any one of the preceding clauses wherein the
compound is of the formula EC1496
##STR00011##
or a pharmaceutically acceptable salt thereof.
[0087] The compound of any one of the preceding clauses wherein the
compound is not of the formula
##STR00012##
or a pharmaceutically acceptable salt thereof.
[0088] The compound of any one of the preceding clauses wherein the
compound is more stable in plasma than the corresponding compound
having fewer unnatural amino acids.
[0089] The compound of any one of the preceding clauses wherein the
compound is more stable in vivo than the corresponding compound
having fewer unnatural amino acids.
[0090] A pharmaceutical composition comprising a compound of any
one of the preceding clauses in combination with one or more
carriers, diluents, or excipients, or a combination thereof.
[0091] A unit dose or unit dosage form composition comprising a
therapeutically effective amount of one or more compounds of any
one of the preceding clauses, optionally in combination with one or
more carriers, diluents, or excipients, or a combination
thereof.
[0092] A method for treating cancer or inflammation in a host
animal, the method comprising the step of administering to the host
animal a composition comprising a therapeutically effective amount
of one or more compounds of any one of the preceding clauses; or a
pharmaceutical composition comprising one or more compounds of any
one of the preceding clauses, optionally further comprising one or
more carriers, diluents, or excipients, or a combination
thereof.
[0093] A method for treating drug resistant cancer in a host
animal, the method comprising the step of administering to the host
animal a composition comprising a therapeutically effective amount
of one or more compounds of any one of the preceding clauses; or a
pharmaceutical composition comprising one or more compounds of any
one of the preceding clauses, optionally further comprising one or
more carriers, diluents, or excipients, or a combination
thereof.
[0094] The method of any one of the preceding clauses wherein the
cancer is an ovarian cancer.
[0095] The method of any one of the preceding clauses wherein the
cancer is a drug resistant ovarian cancer.
[0096] The method of any one of the preceding clauses wherein the
cancer is a platinum resistant ovarian cancer, such as NCI/ADR-RES
or NCI/ADR-RES related ovarian cancer.
[0097] The method of any one of the preceding clauses wherein the
cancer is a platinum resistant ovarian cancer, such as IGROVCDDP or
IGROVCDDP related ovarian cancer.
[0098] The method of any one of the preceding clauses wherein the
cancer is a cisplatin resistant ovarian cancer.
[0099] The method of any one of the preceding clauses wherein the
cancer is a breast cancer.
[0100] The method of any one of the preceding clauses wherein the
cancer is a drug resistant breast cancer.
[0101] The method of any one of the preceding clauses wherein the
cancer is a triple negative breast cancer, such as MDA-MB-231 or
MDA-MB-231 related breast cancer.
[0102] An intermediate for preparing a compound of claim 1 of the
formula
##STR00013##
or a pharmaceutically acceptable salt thereof, wherein L is a
leaving group.
[0103] An intermediate for preparing a compound of claim 1 of the
formula
##STR00014##
or a pharmaceutically acceptable salt thereof, wherein M is
hydrogen or a cation.
[0104] An intermediate for preparing a compound of claim 1 of the
formula
##STR00015##
or a pharmaceutically acceptable salt thereof, wherein L is a
leaving group.
[0105] The compounds described herein can be used for both human
clinical medicine and veterinary applications. Thus, the host
animal harboring the population of pathogenic cells and treated
with the compounds described herein can be human or, in the case of
veterinary applications, can be a laboratory, agricultural,
domestic, or wild animal. The present invention can be applied to
host animals including, but not limited to, humans, laboratory
animals such rodents (e.g., mice, rats, hamsters, etc.), rabbits,
monkeys, chimpanzees, domestic animals such as dogs, cats, and
rabbits, agricultural animals such as cows, horses, pigs, sheep,
goats, and wild animals in captivity such as bears, pandas, lions,
tigers, leopards, elephants, zebras, giraffes, gorillas, dolphins,
and whales.
[0106] The invention is applicable to populations of pathogenic
cells that cause a variety of pathologies in these host animals. In
accordance with the invention "pathogenic cells" means cancer
cells, infectious agents such as bacteria and viruses, bacteria- or
virus-infected cells, activated macrophages capable of causing a
disease state, and any other type of pathogenic cells that uniquely
express, preferentially express, or overexpress vitamin receptors
or receptors that bind analogs or derivatives of vitamins.
Pathogenic cells can also include any cells causing a disease state
for which treatment with the compounds described herein results in
reduction of the symptoms of the disease. For example, the
pathogenic cells can be host cells that are pathogenic under some
circumstances such as cells of the immune system that are
responsible for graft versus host disease, but not pathogenic under
other circumstances.
[0107] Thus, the population of pathogenic cells can be a cancer
cell population that is tumorigenic, including benign tumors and
malignant tumors, or it can be non-tumorigenic. The cancer cell
population can arise spontaneously or by such processes as
mutations present in the germline of the host animal or somatic
mutations, or it can be chemically-, virally-, or
radiation-induced. The invention can be utilized to treat such
cancers as carcinomas, sarcomas, lymphomas, Hodgekin's disease,
melanomas, mesotheliomas, Burkitt's lymphoma, nasopharyngeal
carcinomas, leukemias, and myelomas. The cancer cell population can
include, but is not limited to, oral, thyroid, endocrine, skin,
gastric, esophageal, laryngeal, pancreatic, colon, bladder, bone,
ovarian, cervical, uterine, breast, testicular, prostate, rectal,
kidney, liver, and lung cancers.
[0108] In any of the embodiments described herein heteroatom
linkers can be --NR.sup.1R.sup.2--, oxygen, sulfur, and the
formulae --(NHR.sup.1NHR.sup.2)--, --SO--, --(SO.sub.2)--, and
--N(R.sup.3)O--, wherein R.sup.1, R.sup.2, and R.sup.3 are each
independently selected from hydrogen, alkyl, aryl, arylalkyl,
substituted aryl, substituted arylalkyl, heteroaryl, substituted
heteroaryl, and alkoxyalkyl.
[0109] The releasable linkers can be methylene, 1-alkoxyalkylene,
1-alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl,
1-alkoxycycloalkylenecarbonyl, carbonylarylcarbonyl,
carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl,
haloalkylenecarbonyl, alkylene(dialkylsilyl),
alkylene(alkylarylsilyl), alkylene(diarylsilyl),
(dialkylsilyl)aryl, (alkylarylsilyl)aryl, (diarylsilyl)aryl,
oxycarbonyloxy, oxycarbonyloxyalkyl, sulfonyloxy, oxysulfonylalkyl,
iminoalkylidenyl, carbonylalkylideniminyl, iminocycloalkylidenyl,
carbonylcycloalkylideniminyl, alkylenethio, alkylenearylthio, and
carbonylalkylthio, wherein each of the releasable linkers is
optionally substituted with a substituent X.sup.2, as defined
below.
[0110] In any of the embodiments described herein, the heteroatom
linker can be oxygen, and the releasable linkers can be methylene,
1-alkoxyalkylene, 1-alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl,
and 1-alkoxycycloalkylenecarbonyl, wherein each of the releasable
linkers is optionally substituted with a substituent X.sup.2, as
defined below, and the releasable linker is bonded to the oxygen to
form an acetal or ketal. Alternatively, the heteroatom linker can
be oxygen, and the releasable linker can be methylene, wherein the
methylene is substituted with an optionally-substituted aryl, and
the releasable linker is bonded to the oxygen to form an acetal or
ketal. Further, the heteroatom linker can be oxygen, and the
releasable linker can be sulfonylalkyl, and the releasable linker
is bonded to the oxygen to form an alkylsulfonate.
[0111] In another embodiment of the above releasable linker
embodiment, the heteroatom linker can be nitrogen, and the
releasable linkers can be iminoalkylidenyl,
carbonylalkylideniminyl, iminocycloalkylidenyl, and
carbonylcycloalkylideniminyl, wherein each of the releasable
linkers is optionally substituted with a substituent X.sup.2, as
defined below, and the releasable linker is bonded to the nitrogen
to form an hydrazone. In an alternate configuration, the hydrazone
may be acylated with a carboxylic acid derivative, an orthoformate
derivative, or a carbamoyl derivative to form various acylhydrazone
releasable linkers.
[0112] Alternatively, the heteroatom linker can be oxygen, and the
releasable linkers can be alkylene(dialkylsilyl),
alkylene(alkylarylsilyl), alkylene(diarylsilyl),
(dialkylsilyl)aryl, (alkylarylsilyl)aryl, and (diarylsilyl)aryl,
wherein each of the releasable linkers is optionally substituted
with a substituent X.sup.2, as defined below, and the releasable
linker is bonded to the oxygen to form a silanol.
[0113] In the above releasable linker embodiment, the drug can
include a nitrogen atom, the heteroatom linker can be nitrogen, and
the releasable linkers can be carbonylarylcarbonyl,
carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl,
and the releasable linker can be bonded to the heteroatom nitrogen
to form an amide, and also bonded to the drug nitrogen to form an
amide.
[0114] In the above releasable linker embodiment, the drug can
include an oxygen atom, the heteroatom linker can be nitrogen, and
the releasable linkers can be carbonylarylcarbonyl,
carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl,
and the releasable linker can be bonded to the heteroatom linker
nitrogen to form an amide, and also bonded to the drug oxygen to
form an ester.
[0115] The substituents X.sup.2 can be alkyl, alkoxy, alkoxyalkyl,
hydroxy, hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl,
dialkylaminoalkyl, halo, haloalkyl, sulfhydrylalkyl,
alkylthioalkyl, aryl, substituted aryl, arylalkyl, substituted
arylalkyl, heteroaryl, substituted heteroaryl, carboxy,
carboxyalkyl, alkyl carboxylate, alkyl alkanoate, guanidinoalkyl,
R.sup.4-carbonyl, R.sup.5-carbonylalkyl, R.sup.6-acylamino, and
R.sup.7-acylaminoalkyl, wherein R.sup.4 and R.sup.5 are each
independently selected from amino acids, amino acid derivatives,
and peptides, and wherein R.sup.6 and R.sup.7 are each
independently selected from amino acids, amino acid derivatives,
and peptides.
[0116] In this embodiment the heteroatom linker can be nitrogen,
and the substituent X.sup.2 and the heteroatom linker can be taken
together with the releasable linker to which they are bound to form
an heterocycle.
[0117] The heterocycles can be pyrrolidines, piperidines,
oxazolidines, isoxazolidines, thiazolidines, isothiazolidines,
pyrrolidinones, piperidinones, oxazolidinones, isoxazolidinones,
thiazolidinones, isothiazolidinones, and succinimides.
[0118] In one aspect of the various conjugates described herein,
the polyvalent linker comprises a
3-thiosuccinimid-1-ylalkyloxymethyloxy moiety, where the methyl is
optionally substituted with alkyl or substituted aryl.
[0119] In another aspect, the polyvalent linker comprises a
3-thiosuccinimid-1-ylalkylcarbonyl, where the carbonyl forms an
acylaziridine with the drug, or analog or derivative thereof.
[0120] In another aspect, the polyvalent linker comprises a
1-alkoxycycloalkylenoxy moiety.
[0121] In another aspect, the polyvalent linker comprises an
alkyleneaminocarbonyl(dicarboxylarylene)carboxylate.
[0122] In another aspect, the polyvalent linker comprises a
dithioalkylcarbonylhydrazide, where the hydrazide forms an
hydrazone with the drug, or analog or derivative thereof.
[0123] In another aspect, the polyvalent linker comprises a
3-thiosuccinimid-1-ylalkylcarbonylhydrazide, where the hydrazide
forms a hydrazone with the drug, or analog or derivative
thereof.
[0124] In another aspect, the polyvalent linker comprises a
3-thioalkylsulfonylalkyl(disubstituted silyl)oxy, where the
disubstituted silyl is substituted with alkyl or optionally
substituted aryl.
[0125] In another aspect, the polyvalent linker comprises a
plurality of spacer linkers selected from the group consisting of
the naturally occurring amino acids and stereoisomers thereof.
[0126] In another aspect, the polyvalent linker comprises a
2-dithioalkyloxycarbonyl, where the carbonyl forms a carbonate with
the drug, or analog or derivative thereof.
[0127] In another aspect, the polyvalent linker comprises a
2-dithioarylalkyloxycarbonyl, where the carbonyl forms a carbonate
with the drug, or analog or derivative thereof, and the aryl is
optionally substituted.
[0128] In another aspect, the polyvalent linker comprises a
4-dithioarylalkyloxycarbonyl, where the carbonyl forms a carbonate
with the drug, or analog or derivative thereof, and the aryl is
optionally substituted.
[0129] In another aspect, the polyvalent linker comprises a
3-thiosuccinimid-1-ylalkyloxyalkyloxyalkylidene, where the
alkylidene forms an hydrazone with the drug, or analog or
derivative thereof, each alkyl is independently selected, and the
oxyalkyloxy is optionally substituted with alkyl or optionally
substituted aryl.
[0130] In another aspect, the polyvalent linker comprises a
2-dithioalkyloxycarbonylhydrazide.
[0131] In another aspect, the polyvalent linker comprises a 2- or
3-dithioalkylamino, where the amino forms a vinylogous amide with
the drug, or analog or derivative thereof.
[0132] In another aspect, the polyvalent linker comprises a
2-dithioalkylamino, where the amino forms a vinylogous amide with
the drug, or analog or derivative thereof, and the alkyl is
ethyl.
[0133] In another aspect, the polyvalent linker comprises a 2- or
3-dithioalkylaminocarbonyl, where the carbonyl forms a carbamate
with the drug, or analog or derivative thereof.
[0134] In another aspect, the polyvalent linker comprises a
releasable linker, a spacer linker, and a releasable linker taken
together to form 2-dithioalkylaminocarbonyl, where the carbonyl
forms a carbamate with the drug, or analog or derivative thereof,
and the alkyl is ethyl.
[0135] In another aspect, the polyvalent linker comprises a
2-dithioarylalkyloxycarbonyl, where the carbonyl forms a carbamate
or a carbamoylaziridine with the drug, or analog or derivative
thereof.
[0136] In another aspect, the polyvalent linker comprises a
4-dithioarylalkyloxycarbonyl, where the carbonyl forms a carbamate
or a carbamoylaziridine with the drug, or analog or derivative
thereof.
[0137] In one embodiment, the polyvalent linkers described herein
comprise divalent linkers of formulae (II)
##STR00016##
where n is an integer selected from 1 to about 4; R.sup.a and
R.sup.b are each independently selected from the group consisting
of hydrogen and alkyl, including lower alkyl such as
C.sub.1-C.sub.4 alkyl that are optionally branched; or R.sup.a and
R.sup.b are taken together with the attached carbon atom to form a
carbocyclic ring; R is an optionally substituted alkyl group, an
optionally substituted acyl group, or a suitably selected nitrogen
protecting group; and (*) indicates points of attachment for the
drug, vitamin, imaging agent, diagnostic agent, other bivalent
linkers, or other parts of the conjugate.
[0138] In another embodiment, the polyvalent linkers described
herein comprise divalent linkers of formulae (III)
##STR00017##
where m is an integer selected from 1 to about 4; R is an
optionally substituted alkyl group, an optionally substituted acyl
group, or a suitably selected nitrogen protecting group; and (*)
indicates points of attachment for the drug, vitamin, imaging
agent, diagnostic agent, other bivalent linkers, or other parts of
the conjugate.
[0139] In another embodiment, the polyvalent linkers described
herein comprise divalent linkers of formulae (IV)
##STR00018##
where m is an integer selected from 1 to about 4; R is an
optionally substituted alkyl group, an optionally substituted acyl
group, or a suitably selected nitrogen protecting group; and (*)
indicates points of attachment for the drug, vitamin, imaging
agent, diagnostic agent, other divalent linkers, or other parts of
the conjugate.
[0140] The drug can be any molecule capable of modulating or
otherwise modifying cell function, including pharmaceutically
active compounds. Suitable molecules can include, but are not
limited to: peptides, oligopeptides, retro-inverso oligopeptides,
proteins, protein analogs in which at least one non-peptide linkage
replaces a peptide linkage, apoproteins, glycoproteins, enzymes,
coenzymes, enzyme inhibitors, amino acids and their derivatives,
receptors and other membrane proteins; antigens and antibodies
thereto; haptens and antibodies thereto; hormones, lipids,
phospholipids, liposomes; toxins; antibiotics; analgesics;
bronchodilators; beta-blockers; antimicrobial agents;
antihypertensive agents; cardiovascular agents including
antiarrhythmics, cardiac glycosides, antianginals and vasodilators;
central nervous system agents including stimulants, psychotropics,
antimanics, and depressants; antiviral agents; antihistamines;
cancer drugs including chemotherapeutic agents; tranquilizers;
anti-depressants; H-2 antagonists; anticonvulsants; antinauseants;
prostaglandins and prostaglandin analogs; muscle relaxants;
anti-inflammatory substances; immunosuppressants, stimulants;
decongestants; antiemetics; diuretics; antispasmodics;
antiasthmatics; anti-Parkinson agents; expectorants; cough
suppressants; mucolytics; and mineral and nutritional
additives.
[0141] Further, the drug can be any drug known in the art which is
cytotoxic, enhances tumor permeability, inhibits tumor cell
proliferation, promotes apoptosis, decreases anti-apoptotic
activity in target cells, is used to treat diseases caused by
infectious agents, enhances an endogenous immune response directed
to the pathogenic cells, or is useful for treating a disease state
caused by any type of pathogenic cell. Drugs suitable for use in
accordance with this invention include adrenocorticoids and cortico
steroids, alkylating agents, antiandrogens, antiestrogens,
androgens, aclamycin and aclamycin derivatives, estrogens,
antimetabolites such as cytosine arabinoside, purine analogs,
pyrimidine analogs, and methotrexate, busulfan, carboplatin,
chlorambucil, cisplatin and other platinum compounds, tamoxiphen,
taxol, paclitaxel, paclitaxel derivatives, Taxotere.RTM.,
cyclophosphamide, daunomycin, rhizoxin, T2 toxin, plant alkaloids,
prednisone, hydroxyurea, teniposide, mitomycins, discodermolides,
microtubule inhibitors, epothilones, tubulysins, cyclopropyl
benz[e]indolone, seco-cyclopropyl benz[e]indolone,
O-Ac-seco-cyclopropyl benz[e]indolone, bleomycin and any other
antibiotic, nitrogen mustards, nitrosureas, vinca alkaloids, such
as vincristine, vinblastine, vindesine, vinorelbine and analogs and
derivative thereof such as deacetylvinblastine monohydrazide
(DAVLBH), colchicine, colchicine derivatives, allocolchicine,
thiocolchicine, trityl cysteine, halicondrin B, dolastatins such as
dolastatin 10, amanitins such as .alpha.-amanitin, camptothecin,
irinotecan, and other camptothecin derivatives thereof,
geldanamycin and geldanamycin derivatives, estramustine,
nocodazole, MAP4, colcemid, inflammatory and proinflammatory
agents, peptide and peptidomimetic signal transduction inhibitors,
and any other art-recognized drug or toxin. Other drugs that can be
used in accordance with the invention include rapamycins, such as
sirolimus or everolimus, penicillins, cephalosporins, vancomycin,
erythromycin, clindamycin, rifampin, chloramphenicol,
aminoglycoside antibiotics, gentamicin, amphotericin B, acyclovir,
trifluridine, ganciclovir, zidovudine, amantadine, ribavirin, and
any other art-recognized antimicrobial compound.
[0142] In another embodiment, the drug is selected from a
cryptophycin, bortezomib, thiobortezomib, a tubulysin, aminopterin,
rapamycin, paclitaxel, docetaxel, doxorubicin, daunorubicin,
everolimus, .alpha.-amanatin, verucarin, didemnin B, geldanomycin,
purvalanol A, everolimus, ispinesib, budesonide, dasatinib, an
epothilone, a maytansine, and a tyrosine kinase inhibitor,
including analogs and derivatives of the foregoing. In another
embodiment, the conjugate includes at least two drugs (D) selected
illustratively from a vinca alkaloid, a cryptophycin, bortezomib,
thiobortezomib, a tubulysin, aminopterin, a rapamycin, such as
everolimus or sirolimus, paclitaxel, docetaxel, doxorubicin,
daunorubicin, everolimus, .alpha.-amanatin, verucarin, didemnin B,
geldanomycin, purvalanol A, ispinesib, budesonide, dasatinib, an
epothilone, a maytansine, and a tyrosine kinase inhibitor,
including analogs and derivatives of the foregoing. In one
variation, the drugs (D) are the same. In another variation, the
drugs (D) are different.
[0143] The drug delivery conjugates described herein can be
administered in a combination therapy with any other known drug
whether or not the additional drug is targeted. Illustrative
additional drugs include, but are not limited to, peptides,
oligopeptides, retro-inverso oligopeptides, proteins, protein
analogs in which at least one non-peptide linkage replaces a
peptide linkage, apoproteins, glycoproteins, enzymes, coenzymes,
enzyme inhibitors, amino acids and their derivatives, receptors and
other membrane proteins, antigens and antibodies thereto, haptens
and antibodies thereto, hormones, lipids, phospholipids, liposomes,
toxins, antibiotics, analgesics, bronchodilators, beta-blockers,
antimicrobial agents, antihypertensive agents, cardiovascular
agents including antiarrhythmics, cardiac glycosides, antianginals,
vasodilators, central nervous system agents including stimulants,
psychotropics, antimanics, and depressants, antiviral agents,
antihistamines, cancer drugs including chemotherapeutic agents,
tranquilizers, anti-depressants, H-2 antagonists, anticonvulsants,
antinauseants, prostaglandins and prostaglandin analogs, muscle
relaxants, anti-inflammatory substances, stimulants, decongestants,
antiemetics, diuretics, antispasmodics, antiasthmatics,
anti-Parkinson agents, expectorants, cough suppressants,
mucolytics, and mineral and nutritional additives.
[0144] At least one additional composition comprising a therapeutic
factor can be administered to the host in combination or as an
adjuvant to the above-detailed methodology, to enhance the drug
delivery conjugate-mediated elimination of the population of
pathogenic cells, or more than one additional therapeutic factor
can be administered. The therapeutic factor can be selected from a
compound capable of stimulating an endogenous immune response, a
chemotherapeutic agent, or another therapeutic factor capable of
complementing the efficacy of the administered drug delivery
conjugate. The method of the invention can be performed by
administering to the host, in addition to the above-described
conjugates, compounds or compositions capable of stimulating an
endogenous immune response (e.g. a cytokine) including, but not
limited to, cytokines or immune cell growth factors such as
interleukins 1-18, stem cell factor, basic FGF, EGF, G-CSF, GM-CSF,
FLK-2 ligand, HILDA, MIP-1.alpha., TGF-.alpha., TGF-.beta., M-CSF,
IFN-.alpha., IFN-.beta., IFN-.gamma., soluble CD23, LIF, and
combinations thereof.
[0145] Therapeutically effective combinations of these factors can
be used. In one embodiment, for example, therapeutically effective
amounts of IL-2, for example, in amounts ranging from about 0.1
MIU/m.sup.2/dose/day to about 15 MIU/m.sup.2/dose/day in a multiple
dose daily regimen, and IFN-.alpha., for example, in amounts
ranging from about 0.1 MIU/m.sup.2/dose/day to about 7.5
MIU/m.sup.2/dose/day in a multiple dose daily regimen, can be used
along with the drug delivery conjugates to eliminate, reduce, or
neutralize pathogenic cells in a host animal harboring the
pathogenic cells (MIU=million international units;
m.sup.2=approximate body surface area of an average human). In
another embodiment IL-12 and IFN-.alpha. are used in the
above-described therapeutically effective amounts for interleukins
and interferons, and in yet another embodiment IL-15 and
IFN-.alpha. are used in the above described therapeutically
effective amounts for interleukins and interferons. In an alternate
embodiment IL-2, IFN-.alpha. or IFN-.gamma., and GM-CSF are used in
combination in the above described therapeutically effective
amounts. The invention also contemplates the use of any other
effective combination of cytokines including combinations of other
interleukins and interferons and colony stimulating factors.
[0146] Chemotherapeutic agents, which are, for example, cytotoxic
themselves or can work to enhance tumor permeability, are also
suitable for use in the method of the invention in combination with
the drug delivery conjugates. Such chemotherapeutic agents include
adrenocorticoids and corticosteroids, alkylating agents,
antiandrogens, antiestrogens, androgens, aclamycin and aclamycin
derivatives, estrogens, antimetabolites such as cytosine
arabinoside, purine analogs, pyrimidine analogs, and methotrexate,
busulfan, carboplatin, chlorambucil, cisplatin and other platinum
compounds, tamoxiphen, taxol, paclitaxel, paclitaxel derivatives,
Taxotere.RTM., cyclophosphamide, daunomycin, rhizoxin, T2 toxin,
plant alkaloids, prednisone, hydroxyurea, teniposide, mitomycins,
discodermolides, microtubule inhibitors, epothilones, tubulysin,
cyclopropyl benz[e]indolone, seco-cyclopropyl benz[e]indolone,
O-Ac-seco-cyclopropyl benz[e]indolone, bleomycin and any other
antibiotic, nitrogen mustards, nitrosureas, vincristine,
vinblastine, and analogs and derivative thereof such as
deacetylvinblastine monohydrazide, colchicine, colchicine
derivatives, allocolchicine, thiocolchicine, trityl cysteine,
Halicondrin B, dolastatins such as dolastatin 10, amanitins such as
.alpha.-amanitin, camptothecin, irinotecan, and other camptothecin
derivatives thereof, geldanamycin and geldanamycin derivatives,
estramustine, nocodazole, MAP4, colcemid, inflammatory and
proinflammatory agents, peptide and peptidomimetic signal
transduction inhibitors, and any other art-recognized drug or
toxin. Other drugs that can be used in accordance with the
invention include penicillins, cephalosporins, vancomycin,
erythromycin, clindamycin, rifampin, chloramphenicol,
aminoglycoside antibiotics, gentamicin, amphotericin B, acyclovir,
trifluridine, ganciclovir, zidovudine, amantadine, ribavirin,
maytansines and analogs and derivatives thereof, gemcitabine, and
any other art-recognized antimicrobial compound.
[0147] In another embodiment, amino acid refers to beta, gamma, and
longer amino acids, such as amino acids of the formula:
--N(R)--(CR'R'').sub.q--C(O)--
[0148] where R is hydrogen, alkyl, acyl, or a suitable nitrogen
protecting group, R' and R'' are hydrogen or a substituent, each of
which is independently selected in each occurrence, and q is an
integer such as 1, 2, 3, 4, or 5. Illustratively, R' and/or R''
independently correspond to, but are not limited to, hydrogen or
the side chains present on naturally occurring amino acids, such as
methyl, benzyl, hydroxymethyl, thiomethyl, carboxyl,
carboxylmethyl, guanidinopropyl, and the like, and derivatives and
protected derivatives thereof. The above described formula includes
all stereoisomeric variations. For example, the amino acid may be
selected from asparagine, aspartic acid, cysteine, glutamic acid,
lysine, glutamine, arginine, serine, ornitine, threonine, and the
like.
[0149] In another embodiment, a folate ligand intermediate is
described having the following formula
##STR00019##
wherein m, n, and q are integers that are independently selected
from the range of 0 to about 8; AA is an amino acid, R.sup.1 is
hydrogen, alkyl, or a nitrogen protecting group, and drugs are
optionally attached at the (*) atoms. In one aspect, AA is a
naturally occurring amino acid of either the natural or unnatural
configuration. In another aspect, one or more of AA is a
hydrophilic amino acid. In another aspect, one or more of AA is Asp
and/or Arg. In another aspect, the integer o is 1 or greater. In
another aspect, the integer m is 2 or greater. The drugs, or
analogs or derivatives thereof, and optionally additional linkers
and additional receptor-binding ligands may be connected to the
above formula at the free NH side chains of the
2,.omega.-diaminoalkanoic acid fragments, or at the terminal
carboxylate as indicated by the free valences therein.
[0150] In another embodiment, a folate ligand intermediate is
described having the following formula
##STR00020##
wherein m, n, q, and p are integers that are independently selected
from the range of 0 to about 8; AA is an amino acid, R.sup.1 is
hydrogen, alkyl, or a nitrogen protecting group, and drugs are
optionally attached at the (*) atoms. In one aspect, AA is as a
naturally occurring amino acid of either the natural or unnatural
configuration. In another aspect, one or more of AA is a
hydrophilic amino acid. In another aspect, one or more of AA is Asp
and/or Arg. In another aspect, the integers o and p are 1 or
greater. In another aspect, the integer m is 2 or greater. The
drugs, or analogs or derivatives thereof, and optionally additional
linkers and additional receptor-binding ligands may be connected to
the above formula at the free NH side chains of the
2,.omega.-diaminoalkanoic acid fragments, at the cysteinyl thiol
groups, or at the terminal carboxylate, as indicated by the free
valences therein.
[0151] In another embodiment, a folate ligand intermediate is
described having the following formula
##STR00021##
wherein m, n, q, p, and r are integers that are independently
selected from the range of 0 to about 8; AA is an amino acid,
R.sup.1 is hydrogen, alkyl, or a nitrogen protecting group, and
drugs are optionally attached at the (*) atoms. In one aspect, AA
is as a naturally occurring amino acid of either the natural or
unnatural configuration. In another aspect, one or more of AA is a
hydrophilic amino acid. In another aspect, one or more of AA is Asp
and/or Arg. In another aspect, the integers o, p, and r are 1 or
greater. In another aspect, the integer m is 2 or greater. The
drugs, or analogs or derivatives thereof, and optionally additional
linkers and additional receptor-binding ligands may be connected to
the above formula at the free NH side chains of the
2,.omega.-diaminoalkanoic acid fragments, at the cyteinyl thiol
groups, at the serinyl hydroxy groups, or at the terminal
carboxylate, as indicated by the free valences therein.
[0152] As used herein, tubulysins refer generally to tetrapeptide
compounds of the formula
##STR00022##
and pharmaceutical salts thereof, where
[0153] n is 1-3;
[0154] V is H, OR.sup.2, or halo, and W is H, OR.sup.2, or alkyl,
where R.sup.2 is independently selected in each instance from H,
alkyl, and C(O)R.sup.3, where R.sup.3 is alkyl, cycloalkyl,
alkenyl, aryl, or arylalkyl, each of which is optionally
substituted; providing that R.sup.2 is not H when both V and W are
OR.sup.2; or V and W are taken together with the attached carbon to
form a carbonyl;
[0155] X=H, C.sub.1-4 alkyl, alkenyl, each of which is optionally
substituted, or CH.sub.2QR.sup.9; where Q is --N--, --O--, or
--S--; R.sup.9=H, C.sub.1-4 alkyl, alkenyl, aryl, or C(O)R.sup.10;
and R.sup.10=C.sub.1-6 alkyl, alkenyl, aryl, or heteroaryl, each of
which is optionally substituted;
[0156] Z is alkyl and Y is O; or Z is alkyl or C(O)R.sup.4, and Y
is absent, where R.sup.4 is alkyl, CF.sub.3, or aryl;
[0157] R.sup.1 is H, or R.sup.1 represents 1 to 3 substituents
selected from halo, nitro, carboxylate or a derivative thereof,
cyano, hydroxyl, alkyl, haloalkyl, alkoxy, haloalkoxy, and
OR.sup.6, where R.sup.6 is hydrogen or optionally substituted aryl,
a phenol protecting group, a prodrug moiety, alkyl, arylalkyl,
C(O)R.sup.7, P(O)(OR.sup.8).sub.2, or SO.sub.3R.sup.8, where
R.sup.7 and R.sup.8 are independently selected in each instance
from H, alkyl, alkenyl, cycloalkyl, heterocyclyl, aryl, and
arylalkyl, each of which is optionally substituted, or R.sup.8 is a
metal cation; and
[0158] R is OH or a leaving group, or R forms a carboxylic acid
derivative, such as an acylhydrazide.
[0159] Conjugates of each of the foregoing tubulysins are described
herein. In one variation, Z is methyl. In another variation,
R.sup.1 is H. In another variation, R.sup.1 is OR.sup.6 at C(4),
where R.sup.6 is H, alkyl, or COR.sup.7. In another variation, V is
H, and W is OC(O)R.sup.3. In another variation, X=CH.sub.2QR.sup.9.
In another variation, X=CH.sub.2OR.sup.9. In another variation,
R.sup.9 is alkyl or alkenyl. In another variation, R.sup.9 is
C(O)R.sup.10. In another variation, R.sup.10=optionally substituted
C.sub.1-6 alkyl. In another variation, R.sup.10=C.sub.1-6 alkyl. In
another variation, R forms an acylhydrazide. It is to be understood
that the foregoing description is an explicit description of each
chemically possible combination of variations of the general
tubulysin structure. For example, it is to be understood that the
foregoing description is a description of the variation where Z is
methyl, and R.sup.1 is H; where R.sup.1 is OR.sup.6 at C(4), and
R.sup.6 is H; where Z is methyl, R.sup.1 is OR.sup.6 at C(4),
R.sup.6 is H, and X=CH.sub.2OR.sup.9; and the like.
[0160] Natural tubulysins are generally linear tetrapeptides
consisting of N-methyl pipecolic acid (Mep), isoleucine (Ile), an
unnatural aminoacid called tubuvalin (Tuv), and either an unnatural
aminoacid called tubutyrosine (Tut, an analog of tyrosine) or an
unnatural aminoacid called tubuphenylalanine (Tup, an analog of
phenylalanine). In another embodiment, naturally occurring
tubulysins, and analogs and derivatives thereof, of the following
general formula are described
##STR00023##
and pharmaceutical salts thereof, where R, R.sup.1, and R.sup.10
are as described in the various embodiments herein. Conjugates of
each of the foregoing tubulysins are described herein.
[0161] In another embodiment, conjugates of naturally occurring
tubulysins of the following general formula are described
TABLE-US-00001 ##STR00024## Factor R.sup.10 R.sup.1 A
(CH.sub.3).sub.2CHCH.sub.2 OH B CH.sub.3(CH.sub.2).sub.2 OH C
CH.sub.3CH.sub.2 OH D (CH.sub.3).sub.2CHCH.sub.2 H E
CH.sub.3(CH.sub.2).sub.2 H F CH.sub.2CH.sub.3 H G
(CH.sub.3).sub.2C.dbd.CH OH H CH.sub.3 H I CH.sub.3 OH
and pharmaceutical salts thereof.
[0162] In another embodiment, compounds are described herein where
the conjugate is formed at the terminal carboxylic acid group or
the terminal acylhydrazine group of each of the tybulysins
described herein.
[0163] It is appreciated that the arrangement and/or orientation of
the various hydrophilic linkers may be in a linear or branched
fashion, or both. For example, the hydrophilic linkers may form the
backbone of the linker forming the conjugate between the folate and
the drug, imagining agent, or diagnostic agent. Alternatively, the
hydrophilic portion of the linker may be pendant to or attached to
the backbone of the chain of atoms connecting the binding ligand B
to the drug D. In this latter arrangement, the hydrophilic portion
may be proximal or distal to the backbone chain of atoms.
[0164] In another embodiment, the linker is more or less linear,
and the hydrophilic groups are arranged largely in a series to form
a chain-like linker in the conjugate. Said another way, the
hydrophilic groups form some or all of the backbone of the linker
in this linear embodiment.
[0165] In another embodiment, the linker is branched with
hydrophilic groups. In this branched embodiment, the hydrophilic
groups may be proximal to the backbone or distal to the backbone.
In each of these arrangements, the linker is more spherical or
cylindrical in shape. In one variation, the linker is shaped like a
bottle-brush. In one aspect, the backbone of the linker is formed
by a linear series of amides, and the hydrophilic portion of the
linker is formed by a parallel arrangement of branching side
chains, such as by connecting monosaccharides, sulfonates, and the
like, and derivatives and analogs thereof.
[0166] It is understood that the linker may be neutral or ionizable
under certain conditions, such as physiological conditions
encountered in vivo. For ionizable linkers, under the selected
conditions, the linker may deprotonate to form a negative ion, or
alternatively become protonated to form a positive ion. It is
appreciated that more than one deprotonation or protonation event
may occur. In addition, it is understood that the same linker may
deprotonate and protonate to form inner salts or zwitterionic
compounds.
[0167] In another embodiment, the hydrophilic spacer linkers are
neutral, i.e. under physiological conditions, the linkers do not
significantly protonate nor deprotonate. In another embodiment, the
hydrophilic spacer linkers may be protonated to carry one or more
positive charges. It is understood that the protonation capability
is condition dependent. In one aspect, the conditions are
physiological conditions, and the linker is protonated in vivo. In
another embodiment, the spacers include both regions that are
neutral and regions that may be protonated to carry one or more
positive charges. In another embodiment, the spacers include both
regions that may be deprotonated to carry one or more negative
charges and regions that may be protonated to carry one or more
positive charges. It is understood that in this latter embodiment
that zwitterions or inner salts may be formed.
[0168] In one aspect, the regions of the linkers that may be
deprotonated to carry a negative charge include carboxylic acids,
such as aspartic acid, glutamic acid, and longer chain carboxylic
acid groups, and sulfuric acid esters, such as alkyl esters of
sulfuric acid. In another aspect, the regions of the linkers that
may be protonated to carry a positive charge include amino groups,
such as polyaminoalkylenes including ethylene diamines, propylene
diamines, butylene diamines and the like, and/or heterocycles
including pyrollidines, piperidines, piperazines, and other amino
groups, each of which is optionally substituted. In another
embodiment, the regions of the linkers that are neutral include
poly hydroxyl groups, such as sugars, carbohydrates, saccharides,
inositols, and the like, and/or polyether groups, such as
polyoxyalkylene groups including polyoxyethylene, polyoxypropylene,
and the like.
[0169] In one embodiment, the hydrophilic spacer linkers described
herein include are formed primarily from carbon, hydrogen, and
oxygen, and have a carbon/oxygen ratio of about 3:1 or less, or of
about 2:1 or less. In one aspect, the hydrophilic linkers described
herein include a plurality of ether functional groups. In another
aspect, the hydrophilic linkers described herein include a
plurality of hydroxyl functional groups. Illustrative fragments
that may be used to form such linkers include polyhydroxyl
compounds such as carbohydrates, polyether compounds such as
polyethylene glycol units, and acid groups such as carboxyl and
alkyl sulfuric acids. In one variation, oligoamide spacers, and the
like may also be included in the linker.
[0170] Illustrative carbohydrate spacers include saccharopeptides
as described herein that include both a peptide feature and sugar
feature; glucuronides, which may be incorporated via [2+3] Huisgen
cyclization, also known as click chemistry; .beta.-alkyl
glycosides, such as of 2-deoxyhexapyranoses (2-deoxyglucose,
2-deoxyglucuronide, and the like), and .beta.-alkyl
mannopyranosides. Illustrative PEG groups include those of a
specific length range from about 4 to about 20 PEG groups.
Illustrative alkyl sulfuric acid esters may also be introduced with
click chemistry directly into the backbone. Illustrative oligoamide
spacers include EDTA and DTPA spacers, .beta.-amino acids, and the
like.
[0171] In another embodiment, the hydrophilic spacer linkers
described herein include a polyether, such as the linkers of the
following formulae:
##STR00025##
where m is an integer independently selected in each instance from
1 to about 8; p is an integer selected 1 to about 10; and n is an
integer independently selected in each instance from 1 to about 3.
In one aspect, m is independently in each instance 1 to about 3. In
another aspect, n is 1 in each instance. In another aspect, p is
independently in each instance about 4 to about 6. Illustratively,
the corresponding polypropylene polyethers corresponding to the
foregoing are contemplated herein and may be included in the
conjugates as hydrophilic spacer linkers. In addition, it is
appreciated that mixed polyethylene and polypropylene polyethers
may be included in the conjugates as hydrophilic spacer linkers.
Further, cyclic variations of the foregoing polyether compounds,
such as those that include tetrahydrofuranyl, 1,3-dioxanes,
1,4-dioxanes, and the like are contemplated herein.
[0172] In another illustrative embodiment, the hydrophilic spacer
linkers described herein include a plurality of hydroxyl functional
groups, such as linkers that incorporate monosaccharides,
oligosaccharides, polysaccharides, and the like. It is to be
understood that the polyhydroxyl containing spacer linkers
comprises a plurality of --(CROH)-- groups, where R is hydrogen or
alkyl.
[0173] In another embodiment, the spacer linkers include one or
more of the following fragments:
##STR00026## ##STR00027##
wherein R is H, alkyl, cycloalkyl, or arylalkyl; m is an integer
from 1 to about 3; n is an integer from 1 to about 5, p is an
integer from 1 to about 5, and r is an integer selected from 1 to
about 3. In one aspect, the integer n is 3 or 4. In another aspect,
the integer p is 3 or 4. In another aspect, the integer r is 1.
[0174] In another embodiment, the spacer linker includes one or
more of the following cyclic polyhydroxyl groups:
##STR00028## ##STR00029##
wherein n is an integer from 2 to about 5, p is an integer from 1
to about 5, and r is an integer from 1 to about 4. In one aspect,
the integer n is 3 or 4. In another aspect, the integer p is 3 or
4. In another aspect, the integer r is 2 or 3. It is understood
that all stereochemical forms of such sections of the linkers are
contemplated herein. For example, in the above formula, the section
may be derived from ribose, xylose, glucose, mannose, galactose, or
other sugar and retain the stereochemical arrangements of pendant
hydroxyl and alkyl groups present on those molecules. In addition,
it is to be understood that in the foregoing formulae, various
deoxy compounds are also contemplated. Illustratively, compounds of
the following formulae are contemplated:
##STR00030##
wherein n is equal to or less than r, such as when r is 2 or 3, n
is 1 or 2, or 1, 2, or 3, respectively.
[0175] In another embodiment, the spacer linker includes a
polyhydroxyl compound of the following formula:
##STR00031##
wherein n and r are each an integer selected from 1 to about 3. In
one aspect, the spacer linker includes one or more polyhydroxyl
compounds of the following formulae:
##STR00032##
It is understood that all stereochemical forms of such sections of
the linkers are contemplated herein. For example, in the above
formula, the section may be derived from ribose, xylose, glucose,
mannose, galactose, or other sugar and retain the stereochemical
arrangements of pendant hydroxyl and alkyl groups present on those
molecules.
[0176] In another configuration, the hydrophilic linkers L
described herein include polyhydroxyl groups that are spaced away
from the backbone of the linker. In one embodiment, such
carbohydrate groups or polyhydroxyl groups are connected to the
back bone by a triazole group, forming triazole-linked hydrophilic
spacer linkers. Illustratively, such linkers include fragments of
the following formulae:
##STR00033##
wherein n, m, and r are integers and are each independently
selected in each instance from 1 to about 5. In one illustrative
aspect, m is independently 2 or 3 in each instance. In another
aspect, r is 1 in each instance. In another aspect, n is 1 in each
instance. In one variation, the group connecting the polyhydroxyl
group to the backbone of the linker is a different heteroaryl
group, including but not limited to, pyrrole, pyrazole,
1,2,4-triazole, furan, oxazole, isoxazole, thienyl, thiazole,
isothiazole, oxadiazole, and the like. Similarly, divalent
6-membered ring heteroaryl groups are contemplated. Other
variations of the foregoing illustrative hydrophilic spacer linkers
include oxyalkylene groups, such as the following formulae:
##STR00034##
wherein n and r are integers and are each independently selected in
each instance from 1 to about 5; and p is an integer selected from
1 to about 4.
[0177] In another embodiment, such carbohydrate groups or
polyhydroxyl groups are connected to the back bone by an amide
group, forming amide-linked hydrophilic spacer linkers.
Illustratively, such linkers include fragments of the following
formulae:
##STR00035##
wherein n is an integer selected from 1 to about 3, and m is an
integer selected from 1 to about 22. In one illustrative aspect, n
is 1 or 2. In another illustrative aspect, m is selected from about
6 to about 10, illustratively 8. In one variation, the group
connecting the polyhydroxyl group to the backbone of the linker is
a different functional group, including but not limited to, esters,
ureas, carbamates, acylhydrazones, and the like. Similarly, cyclic
variations are contemplated. Other variations of the foregoing
illustrative hydrophilic spacer linkers include oxyalkylene groups,
such as the following formulae:
##STR00036##
wherein n and r are integers and are each independently selected in
each instance from 1 to about 5; and p is an integer selected from
1 to about 4.
[0178] In another embodiment, the spacer linkers include one or
more of the following fragments:
##STR00037## ##STR00038## ##STR00039##
wherein R is H, alkyl, cycloalkyl, or arylalkyl; m is an
independently selected integer from 1 to about 3; n is an integer
from 1 to about 6, p is an integer from 1 to about 5, and r is an
integer selected from 1 to about 3. In one variation, the integer n
is 3 or 4. In another variation, the integer p is 3 or 4. In
another variation, the integer r is 1.
[0179] In another embodiment, the spacer linkers include one or
more of the following fragments:
##STR00040## ##STR00041## ##STR00042##
wherein m is an independently selected integer from 1 to about 3; n
is an integer from 1 to about 6, p is an integer from 1 to about 5,
and r is an integer selected from 1 to about 3. In one variation,
the integer n is 3 or 4. In another variation, the integer p is 3
or 4. In another variation, the integer r is 1.
[0180] In another embodiment, the spacer linkers include one or
more of the following fragments:
##STR00043## ##STR00044## ##STR00045##
wherein m is an independently selected integer from 1 to about 3; n
is an integer from 1 to about 6, p is an integer from 1 to about 5,
and r is an integer selected from 1 to about 3. In one variation,
the integer n is 3 or 4. In another variation, the integer p is 3
or 4. In another variation, the integer r is 1.
[0181] In another embodiment, the hydrophilic spacer linker is a
combination of backbone and branching side motifs such as is
illustrated by the following formulae
##STR00046##
wherein n is an integer independently selected in each instance
from 0 to about 3. The above formula are intended to represent 4,
5, 6, and even larger membered cyclic sugars. In addition, it is to
be understood that the above formula may be modified to represent
deoxy sugars, where one or more of the hydroxy groups present on
the formulae are replaced by hydrogen, alkyl, or amino. In
addition, it is to be understood that the corresponding carbonyl
compounds are contemplated by the above formulae, where one or more
of the hydroxyl groups is oxidized to the corresponding carbonyl.
In addition, in this illustrative embodiment, the pyranose includes
both carboxyl and amino functional groups and (a) can be inserted
into the backbone and (b) can provide synthetic handles for
branching side chains in variations of this embodiment. Any of the
pendant hydroxyl groups may be used to attach other chemical
fragments, including additional sugars to prepare the corresponding
oligosaccharides. Other variations of this embodiment are also
contemplated, including inserting the pyranose or other sugar into
the backbone at a single carbon, i.e. a spiro arrangement, at a
geminal pair of carbons, and like arrangements. For example, one or
two ends of the linker, or the drug D, or the binding ligand B may
be connected to the sugar to be inserted into the backbone in a
1,1; 1,2; 1,3; 1,4; 2,3, or other arrangement.
[0182] In another embodiment, the hydrophilic spacer linkers
described herein include are formed primarily from carbon,
hydrogen, and nitrogen, and have a carbon/nitrogen ratio of about
3:1 or less, or of about 2:1 or less. In one aspect, the
hydrophilic linkers described herein include a plurality of amino
functional groups.
[0183] In another embodiment, the spacer linkers include one or
more amino groups of the following formulae:
##STR00047##
where n is an integer independently selected in each instance from
1 to about 3. In one aspect, the integer n is independently 1 or 2
in each instance. In another aspect, the integer n is 1 in each
instance.
[0184] In another embodiment, the hydrophilic spacer linker is a
sulfuric acid ester, such as an alkyl ester of sulfuric acid.
Illustratively, the spacer linker is of the following formula:
##STR00048##
where n is an integer independently selected in each instance from
1 to about 3. Illustratively, n is independently 1 or 2 in each
instance.
[0185] It is understood, that in such polyhydroxyl, polyamino,
carboxylic acid, sulfuric acid, and like linkers that include free
hydrogens bound to heteroatoms, one or more of those free hydrogen
atoms may be protected with the appropriate hydroxyl, amino, or
acid protecting group, respectively, or alternatively may be
blocked as the corresponding pro-drugs, the latter of which are
selected for the particular use, such as pro-drugs that release the
parent drug under general or specific physiological conditions.
[0186] In each of the foregoing illustrative examples of linkers L,
there are also included in some cases additional spacer linkers
L.sub.S, and/or additional releasable linkers L.sub.R. Those spacer
linker and releasable linkers also may include asymmetric carbon
atoms. It is to be further understood that the stereochemical
configurations shown herein are merely illustrative, and other
stereochemical configurations are contemplated. For example in one
variation, the corresponding unnatural amino acid configurations
may be included in the conjugated described herein as follows:
##STR00049##
wherein n is an integer from 2 to about 5, p is an integer from 1
to about 5, and r is an integer from 1 to about 4, as described
above.
[0187] It is to be further understood that in the foregoing
embodiments, open positions, such as (*) atoms are locations for
attachment of the binding ligand (B) or the drug (D) to be
delivered. In addition, it is to be understood that such attachment
of either or both of B and A may be direct or through an
intervening linker. Intervening linkers include other spacer
linkers and/or releasable linkers. Illustrative additional spacer
linkers and releasable linkers that are included in the conjugated
described herein are described in U.S. patent application Ser. No.
10/765,335, the disclosure of which is incorporated herein by
reference.
[0188] In one embodiment, the hydrophilic spacer linker comprises
one or more carbohydrate containing or polyhydroxyl group
containing linkers. In another embodiment, the hydrophilic spacer
linker comprises at least three carbohydrate containing or
polyhydroxyl group containing linkers. In another embodiment, the
hydrophilic spacer linker comprises one or more carbohydrate
containing or polyhydroxyl group containing linkers, and one or
more aspartic acids. In another embodiment, the hydrophilic spacer
linker comprises one or more carbohydrate containing or
polyhydroxyl group containing linkers, and one or more glutamic
acids. In another embodiment, the hydrophilic spacer linker
comprises one or more carbohydrate containing or polyhydroxyl group
containing linkers, one or more glutamic acids, one or more
aspartic acids, and one or more beta amino alanines. In a series of
variations, in each of the foregoing embodiments, the hydrophilic
spacer linker also includes one or more cysteines. In another
series of variations, in each of the foregoing embodiments, the
hydrophilic spacer linker also includes at least one arginine.
[0189] In another embodiment, the hydrophilic spacer linker
comprises one or more divalent 1,4-piperazines that are included in
the chain of atoms connecting at least one of the binding ligands
(L) with at least one of the drugs (D). In one variation, the
hydrophilic spacer linker includes one or more carbohydrate
containing or polyhydroxyl group containing linkers.
[0190] In another variation, the hydrophilic spacer linker includes
one or more carbohydrate containing or polyhydroxyl group
containing linkers and one or more aspartic acids. In another
variation, the hydrophilic spacer linker includes one or more
carbohydrate containing or polyhydroxyl group containing linkers
and one or more glutamic acids. In a series of variations, in each
of the foregoing embodiments, the hydrophilic spacer linker also
includes one or more cysteines. In another series of variations, in
each of the foregoing embodiments, the hydrophilic spacer linker
also includes at least one arginine.
[0191] In another embodiment, the hydrophilic spacer linker
comprises one or more oligoamide hydrophilic spacers, such as but
not limited to aminoethylpiperazinylacetamide.
[0192] In another embodiment, the hydrophilic spacer linker
comprises one or more triazole linked carbohydrate containing or
polyhydroxyl group containing linkers. In another embodiment, the
hydrophilic spacer linker comprises one or more amide linked
carbohydrate containing or polyhydroxyl group containing linkers.
In another embodiment, the hydrophilic spacer linker comprises one
or more PEG groups and one or more cysteines. In another
embodiment, the hydrophilic spacer linker comprises one or more
EDTE derivatives.
[0193] Illustrative embodiments of vitamin analogs and/or
derivatives include folate and analogs and derivatives of folate
such as folinic acid, pteropolyglutamic acid, and folate
receptor-binding pteridines such as tetrahydropterins,
dihydrofolates, tetrahydrofolates, and their deaza and dideaza
analogs. The terms "deaza" and "dideaza" analogs refer to the
art-recognized analogs having a carbon atom substituted for one or
two nitrogen atoms in the naturally occurring folic acid structure,
or analog or derivative thereof. For example, the deaza analogs
include the 1-deaza, 3-deaza, 5-deaza, 8-deaza, and 10-deaza
analogs of folate, folinic acid, pteropolyglutamic acid, and folate
receptor-binding pteridines such as tetrahydropterins,
dihydrofolates, and tetrahydrofolates. The dideaza analogs include,
for example, 1,5-dideaza, 5,10-dideaza, 8,10-dideaza, and
5,8-dideaza analogs of folate, folinic acid, pteropolyglutamic
acid, and folate receptor-binding pteridines such as
tetrahydropterins, dihydrofolates, and tetrahydrofolates. Other
folates useful as complex forming ligands for this invention are
the folate receptor-binding analogs aminopterin, amethopterin (also
known as methotrexate), N.sup.10-methylfolate,
2-deamino-hydroxyfolate, deaza analogs such as 1-deazamethopterin
or 3-deazamethopterin, and
3',5'-dichloro-4-amino-4-deoxy-N.sup.10-methylpteroylglutamic acid
(dichloromethotrexate). The foregoing folic acid analogs and/or
derivatives are conventionally termed "folates," reflecting their
ability to bind with folate-receptors, and such ligands when
conjugated with exogenous molecules are effective to enhance
transmembrane transport, such as via folate-mediated endocytosis as
described herein.
[0194] In another embodiment, the drug has the formula
##STR00050##
wherein
[0195] Y.sup.A is OR.sup.C or OCH.sub.2CH.sub.2OR.sup.C;
[0196] one of R.sup.A, R.sup.B, or R.sup.C is a bond connected to
L; and
[0197] the other two of R.sup.A, R.sup.B, and R.sup.C are
independently selected in each case from the group consisting of
hydrogen, optionally substituted heteroalkyl, prodrug forming
group, and C(O)R.sup.D, where R.sup.D is in each instance
independently selected from the group consisting of hydrogen, and
alkyl, alkenyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl,
arylalkyl, heteroaryl, and heteroarylalkyl, each of which is
optionally substituted is described.
[0198] In another embodiment, the compound of any of the embodiment
described herein wherein L comprises a divalent linker of the
formula
##STR00051##
[0199] wherein * indicates the point of attachment to a folate and
** indicates the point of attachment to a drug; and F and G are
each independently 1, 2, 3 or 4 are described.
[0200] In another embodiment, the of any one of the embodiments
described herein wherein L is a linker comprises a divalent linker
of the formula
##STR00052##
[0201] wherein *, **, *** each indicate points of attachment to the
folate receptor binding moiety B, and the one or more drugs D. It
is to be understood that when there are fewer drugs, *, **, *** are
substituted with hydrogen or a heteroatom. F and G are each
independently 1, 2, 3 or 4; and W.sup.1 is NH or O is described. In
another aspect, m.sup.1 is 0 or 1.
[0202] In another embodiment, the method or pharmaceutical
composition of any one of the preceding embodiments wherein the
disease is selected from the group consisting of arthritis,
including rheumatoid arthritis and osteoarthritis,
glomerulonephritis, proliferative retinopathy, restenosis,
ulcerative colitis, Crohn's disease, fibromyalgia, psoriasis and
other inflammations of the skin, osteomyelitis, Sjogren's syndrome,
multiple sclerosis, diabetes, atherosclerosis, pulmonary fibrosis,
lupus erythematosus, sarcoidosis, systemic sclerosis, organ
transplant rejection (GVHD) and chronic inflammations is
described.
[0203] The compounds described herein may contain one or more
chiral centers, or may otherwise be capable of existing as multiple
stereoisomers. It is to be understood that in one embodiment, the
invention described herein is not limited to any particular
sterochemical requirement, and that the compounds, and
compositions, methods, uses, and medicaments that include them may
be optically pure, or may be any of a variety of stereoisomeric
mixtures, including racemic and other mixtures of enantiomers,
other mixtures of diastereomers, and the like. It is also to be
understood that such mixtures of stereoisomers may include a single
stereochemical configuration at one or more chiral centers, while
including mixtures of stereochemical configuration at one or more
other chiral centers.
[0204] Similarly, the compounds described herein may be include
geometric centers, such as cis, trans, E, and Z double bonds. It is
to be understood that in another embodiment, the invention
described herein is not limited to any particular geometric isomer
requirement, and that the compounds, and compositions, methods,
uses, and medicaments that include them may be pure, or may be any
of a variety of geometric isomer mixtures. It is also to be
understood that such mixtures of geometric isomers may include a
single configuration at one or more double bonds, while including
mixtures of geometry at one or more other double bonds.
[0205] As used herein, the term "alkyl" includes a chain of carbon
atoms, which is optionally branched. As used herein, the term
"alkenyl" and "alkynyl" includes a chain of carbon atoms, which is
optionally branched, and includes at least one double bond or
triple bond, respectively. It is to be understood that alkynyl may
also include one or more double bonds. It is to be further
understood that in certain embodiments, alkyl is advantageously of
limited length, including C.sub.1-C.sub.24, C.sub.1-C.sub.12,
C.sub.1-C.sub.8, C.sub.1-C.sub.6, and C.sub.1-C.sub.4.
Illustratively, such particularly limited length alkyl groups,
including C.sub.1-C.sub.8, C.sub.1-C.sub.6, and C.sub.1-C.sub.4 may
be referred to as lower alkyl. It is to be further understood that
in certain embodiments alkenyl and/or alkynyl may each be
advantageously of limited length, including C.sub.2-C.sub.24,
C.sub.2-C.sub.12, C.sub.2-C.sub.8, C.sub.2-C.sub.6, and
C.sub.2-C.sub.4. Illustratively, such particularly limited length
alkenyl and/or alkynyl groups, including C.sub.2-C.sub.8,
C.sub.2-C.sub.6, and C.sub.2-C.sub.4 may be referred to as lower
alkenyl and/or alkynyl. It is appreciated herein that shorter
alkyl, alkenyl, and/or alkynyl groups may add less lipophilicity to
the compound and accordingly will have different pharmacokinetic
behavior. In embodiments of the invention described herein, it is
to be understood, in each case, that the recitation of alkyl refers
to alkyl as defined herein, and optionally lower alkyl. In
embodiments of the invention described herein, it is to be
understood, in each case, that the recitation of alkenyl refers to
alkenyl as defined herein, and optionally lower alkenyl. In
embodiments of the invention described herein, it is to be
understood, in each case, that the recitation of alkynyl refers to
alkynyl as defined herein, and optionally lower alkynyl.
Illustrative alkyl, alkenyl, and alkynyl groups are, but not
limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,
sec-butyl, tert-butyl, pentyl, 2-pentyl, 3-pentyl, neopentyl,
hexyl, heptyl, octyl, and the like, and the corresponding groups
containing one or more double and/or triple bonds, or a combination
thereof.
[0206] As used herein, the term "alkylene" includes a divalent
chain of carbon atoms, which is optionally branched. As used
herein, the term "alkenylene" and "alkynylene" includes a divalent
chain of carbon atoms, which is optionally branched, and includes
at least one double bond or triple bond, respectively. It is to be
understood that alkynylene may also include one or more double
bonds. It is to be further understood that in certain embodiments,
alkylene is advantageously of limited length, including
C.sub.1-C.sub.24, C.sub.1-C.sub.12, C.sub.1-C.sub.8,
C.sub.1-C.sub.6, and C.sub.1-C.sub.4. Illustratively, such
particularly limited length alkylene groups, including
C.sub.1-C.sub.8, C.sub.1-C.sub.6, and C.sub.1-C.sub.4 may be
referred to as lower alkylene. It is to be further understood that
in certain embodiments alkenylene and/or alkynylene may each be
advantageously of limited length, including C.sub.2-C.sub.24,
C.sub.2-C.sub.12, C.sub.2-C.sub.8, C.sub.2-C.sub.6, and
C.sub.2-C.sub.4. Illustratively, such particularly limited length
alkenylene and/or alkynylene groups, including C.sub.2-C.sub.8,
C.sub.2-C.sub.6, and C.sub.2-C.sub.4 may be referred to as lower
alkenylene and/or alkynylene. It is appreciated herein that shorter
alkylene, alkenylene, and/or alkynylene groups may add less
lipophilicity to the compound and accordingly will have different
pharmacokinetic behavior. In embodiments of the invention described
herein, it is to be understood, in each case, that the recitation
of alkylene, alkenylene, and alkynylene refers to alkylene,
alkenylene, and alkynylene as defined herein, and optionally lower
alkylene, alkenylene, and alkynylene. Illustrative alkyl groups
are, but not limited to, methylene, ethylene, n-propylene,
isopropylene, n-butylene, isobutylene, sec-butylene, pentylene,
1,2-pentylene, 1,3-pentylene, hexylene, heptylene, octylene, and
the like.
[0207] As used herein, the term "cycloalkyl" includes a chain of
carbon atoms, which is optionally branched, where at least a
portion of the chain in cyclic. It is to be understood that
cycloalkylalkyl is a subset of cycloalkyl. It is to be understood
that cycloalkyl may be polycyclic. Illustrative cycloalkyl include,
but are not limited to, cyclopropyl, cyclopentyl, cyclohexyl,
2-methylcyclopropyl, cyclopentyleth-2-yl, adamantyl, and the like.
As used herein, the term "cycloalkenyl" includes a chain of carbon
atoms, which is optionally branched, and includes at least one
double bond, where at least a portion of the chain in cyclic. It is
to be understood that the one or more double bonds may be in the
cyclic portion of cycloalkenyl and/or the non-cyclic portion of
cycloalkenyl. It is to be understood that cycloalkenylalkyl and
cycloalkylalkenyl are each subsets of cycloalkenyl. It is to be
understood that cycloalkyl may be polycyclic. Illustrative
cycloalkenyl include, but are not limited to, cyclopentenyl,
cyclohexylethen-2-yl, cycloheptenylpropenyl, and the like. It is to
be further understood that chain forming cycloalkyl and/or
cycloalkenyl is advantageously of limited length, including
C.sub.3-C.sub.24, C.sub.3-C.sub.12, C.sub.3-C.sub.8,
C.sub.3-C.sub.6, and C.sub.5-C.sub.6. It is appreciated herein that
shorter alkyl and/or alkenyl chains forming cycloalkyl and/or
cycloalkenyl, respectively, may add less lipophilicity to the
compound and accordingly will have different pharmacokinetic
behavior.
[0208] As used herein, the term "heteroalkyl" includes a chain of
atoms that includes both carbon and at least one heteroatom, and is
optionally branched. Illustrative heteroatoms include nitrogen,
oxygen, and sulfur. In certain variations, illustrative heteroatoms
also include phosphorus, and selenium. As used herein, the term
"cycloheteroalkyl" including heterocyclyl and heterocycle, includes
a chain of atoms that includes both carbon and at least one
heteroatom, such as heteroalkyl, and is optionally branched, where
at least a portion of the chain is cyclic. Illustrative heteroatoms
include nitrogen, oxygen, and sulfur. In certain variations,
illustrative heteroatoms also include phosphorus, and selenium.
Illustrative cycloheteroalkyl include, but are not limited to,
tetrahydrofuryl, pyrrolidinyl, tetrahydropyranyl, piperidinyl,
morpholinyl, piperazinyl, homopiperazinyl, quinuclidinyl, and the
like.
[0209] As used herein, the term "aryl" includes monocyclic and
polycyclic aromatic carbocyclic groups, each of which may be
optionally substituted. Illustrative aromatic carbocyclic groups
described herein include, but are not limited to, phenyl, naphthyl,
and the like. As used herein, the term "heteroaryl" includes
aromatic heterocyclic groups, each of which may be optionally
substituted. Illustrative aromatic heterocyclic groups include, but
are not limited to, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl,
tetrazinyl, quinolinyl, quinazolinyl, quinoxalinyl, thienyl,
pyrazolyl, imidazolyl, oxazolyl, thiazolyl, isoxazolyl,
isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, benzimidazolyl,
benzoxazolyl, benzthiazolyl, benzisoxazolyl, benzisothiazolyl, and
the like.
[0210] As used herein, the term "amino" includes the group
NH.sub.2, alkylamino, and dialkylamino, where the two alkyl groups
in dialkylamino may be the same or different, i.e. alkylalkylamino.
Illustratively, amino includes methylamino, ethylamino,
dimethylamino, methylethylamino, and the like. In addition, it is
to be understood that when amino modifies or is modified by another
term, such as aminoalkyl, or acylamino, the above variations of the
term amino are included therein. Illustratively, aminoalkyl
includes H.sub.2N-alkyl, methylaminoalkyl, ethylaminoalkyl,
dimethylaminoalkyl, methylethylaminoalkyl, and the like.
Illustratively, acylamino includes acylmethylamino, acylethylamino,
and the like.
[0211] As used herein, the term "amino and derivatives thereof"
includes amino as described herein, and alkylamino, alkenylamino,
alkynylamino, heteroalkylamino, heteroalkenylamino,
heteroalkynylamino, cycloalkylamino, cycloalkenylamino,
cycloheteroalkylamino, cycloheteroalkenylamino, arylamino,
arylalkylamino, arylalkenylamino, arylalkynylamino,
heteroarylamino, heteroarylalkylamino, heteroarylalkenylamino,
heteroarylalkynylamino, acylamino, and the like, each of which is
optionally substituted. The term "amino derivative" also includes
urea, carbamate, and the like.
[0212] As used herein, the term "hydroxy and derivatives thereof"
includes OH, and alkyloxy, alkenyloxy, alkynyloxy, heteroalkyloxy,
heteroalkenyloxy, heteroalkynyloxy, cycloalkyloxy, cycloalkenyloxy,
cycloheteroalkyloxy, cycloheteroalkenyloxy, aryloxy, arylalkyloxy,
arylalkenyloxy, arylalkynyloxy, heteroaryloxy, heteroarylalkyloxy,
heteroarylalkenyloxy, heteroarylalkynyloxy, acyloxy, and the like,
each of which is optionally substituted. The term "hydroxy
derivative" also includes carbamate, and the like.
[0213] As used herein, the term "thio and derivatives thereof"
includes SH, and alkylthio, alkenylthio, alkynylthio,
heteroalkylthio, heteroalkenylthio, heteroalkynylthio,
cycloalkylthio, cycloalkenylthio, cycloheteroalkylthio,
cycloheteroalkenylthio, arylthio, arylalkylthio, arylalkenylthio,
arylalkynylthio, heteroarylthio, heteroarylalkylthio,
heteroarylalkenylthio, heteroarylalkynylthio, acylthio, and the
like, each of which is optionally substituted. The term "thio
derivative" also includes thiocarbamate, and the like.
[0214] As used herein, the term "acyl" includes formyl, and
alkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl,
heteroalkylcarbonyl, heteroalkenylcarbonyl, heteroalkynylcarbonyl,
cycloalkylcarbonyl, cycloalkenylcarbonyl, cycloheteroalkylcarbonyl,
cycloheteroalkenylcarbonyl, arylcarbonyl, arylalkylcarbonyl,
arylalkenylcarbonyl, arylalkynylcarbonyl, heteroarylcarbonyl,
heteroarylalkylcarbonyl, heteroarylalkenylcarbonyl,
heteroarylalkynylcarbonyl, acylcarbonyl, and the like, each of
which is optionally substituted.
[0215] As used herein, the term "carbonyl and derivatives thereof"
includes the group C(O), C(S), C(NH) and substituted amino
derivatives thereof.
[0216] As used herein, the term "carboxylic acid and derivatives
thereof" includes the group CO.sub.2H and salts thereof, and esters
and amides thereof, and CN.
[0217] The term "optionally substituted" as used herein includes
the replacement of hydrogen atoms with other functional groups on
the radical that is optionally substituted. Such other functional
groups illustratively include, but are not limited to, amino,
hydroxyl, halo, thiol, alkyl, haloalkyl, heteroalkyl, aryl,
arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl,
heteroarylheteroalkyl, nitro, sulfonic acids and derivatives
thereof, carboxylic acids and derivatives thereof, and the like.
Illustratively, any of amino, hydroxyl, thiol, alkyl, haloalkyl,
heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl,
heteroarylalkyl, heteroarylheteroalkyl, and/or sulfonic acid is
optionally substituted.
[0218] As used herein, the terms "optionally substituted aryl" and
"optionally substituted heteroaryl" include the replacement of
hydrogen atoms with other functional groups on the aryl or
heteroaryl that is optionally substituted. Such other functional
groups illustratively include, but are not limited to, amino,
hydroxy, halo, thio, alkyl, haloalkyl, heteroalkyl, aryl,
arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl,
heteroarylheteroalkyl, nitro, sulfonic acids and derivatives
thereof, carboxylic acids and derivatives thereof, and the like.
Illustratively, any of amino, hydroxy, thio, alkyl, haloalkyl,
heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl,
heteroarylalkyl, heteroarylheteroalkyl, and/or sulfonic acid is
optionally substituted.
[0219] Illustrative substituents include, but are not limited to, a
radical --(CH.sub.2).sub.xZ.sup.X, where x is an integer from 0-6
and Z.sup.X is selected from halogen, hydroxy, alkanoyloxy,
including C.sub.1-C.sub.6 alkanoyloxy, optionally substituted
aroyloxy, alkyl, including C.sub.1-C.sub.6 alkyl, alkoxy, including
C.sub.1-C.sub.6 alkoxy, cycloalkyl, including C.sub.3-C.sub.8
cycloalkyl, cycloalkoxy, including C.sub.3-C.sub.8 cycloalkoxy,
alkenyl, including C.sub.2-C.sub.6 alkenyl, alkynyl, including
C.sub.2-C.sub.6 alkynyl, haloalkyl, including C.sub.1-C.sub.6
haloalkyl, haloalkoxy, including C.sub.1-C.sub.6 haloalkoxy,
halocycloalkyl, including C.sub.3-C.sub.8 halocycloalkyl,
halocycloalkoxy, including C.sub.3-C.sub.8 halocycloalkoxy, amino,
C.sub.1-C.sub.6 alkylamino, (C.sub.1-C.sub.6 alkyl)(C.sub.1-C.sub.6
alkyl)amino, alkylcarbonylamino, N--(C.sub.1-C.sub.6
alkyl)alkylcarbonylamino, aminoalkyl, C.sub.1-C.sub.6
alkylaminoalkyl, (C.sub.1-C.sub.6 alkyl)(C.sub.1-C.sub.6
alkyl)aminoalkyl, alkylcarbonylaminoalkyl, N--(C.sub.1-C.sub.6
alkyl)alkylcarbonylaminoalkyl, cyano, and nitro; or Z.sup.X is
selected from --CO.sub.2R.sup.4 and --CONR.sup.5R.sup.6, where
R.sup.4, R.sup.5, and R.sup.6 are each independently selected in
each occurrence from hydrogen, C.sub.1-C.sub.6 alkyl,
aryl-C.sub.1-C.sub.6 alkyl, and heteroaryl-C.sub.1-C.sub.6
alkyl.
[0220] As used herein, the term "composition" generally refers to
any product comprising the specified ingredients in the specified
amounts, as well as any product which results, directly or
indirectly, from combinations of the specified ingredients in the
specified amounts. It is to be understood that the compositions
described herein may be prepared from isolated compounds described
herein or from salts, solutions, hydrates, solvates, and other
forms of the compounds described herein. It is also to be
understood that the compositions may be prepared from various
amorphous, non-amorphous, partially crystalline, crystalline,
and/or other morphological forms of the compounds described herein.
It is also to be understood that the compositions may be prepared
from various hydrates and/or solvates of the compounds described
herein. Accordingly, such pharmaceutical compositions that recite
compounds described herein are to be understood to include each of,
or any combination of, the various morphological forms and/or
solvate or hydrate forms of the compounds described herein.
Illustratively, compositions may include one or more carriers,
diluents, and/or excipients. The compounds described herein, or
compositions containing them, may be formulated in a
therapeutically effective amount in any conventional dosage forms
appropriate for the methods described herein. The compounds
described herein, or compositions containing them, including such
formulations, may be administered by a wide variety of conventional
routes for the methods described herein, and in a wide variety of
dosage formats, utilizing known procedures (see generally,
Remington: The Science and Practice of Pharmacy, (21.sup.st ed.,
2005)).
[0221] The term "therapeutically effective amount" as used herein,
refers to that amount of active compound or pharmaceutical agent
that elicits the biological or medicinal response in a tissue
system, animal or human that is being sought by a researcher,
veterinarian, medical doctor or other clinician, which includes
alleviation of the symptoms of the disease or disorder being
treated. In one aspect, the therapeutically effective amount is
that which may treat or alleviate the disease or symptoms of the
disease at a reasonable benefit/risk ratio applicable to any
medical treatment. However, it is to be understood that the total
daily usage of the compounds and compositions described herein may
be decided by the attending physician within the scope of sound
medical judgment. The specific therapeutically-effective dose level
for any particular patient will depend upon a variety of factors,
including the disorder being treated and the severity of the
disorder; activity of the specific compound employed; the specific
composition employed; the age, body weight, general health, gender
and diet of the patient: the time of administration, route of
administration, and rate of excretion of the specific compound
employed; the duration of the treatment; drugs used in combination
or coincidentally with the specific compound employed; and like
factors well known to the researcher, veterinarian, medical doctor
or other clinician of ordinary skill
[0222] The term "administering" as used herein includes all means
of introducing the compounds and compositions described herein to
the patient, including, but are not limited to, oral (po),
intravenous (iv), intramuscular (im), subcutaneous (sc),
transdermal, inhalation, buccal, ocular, sublingual, vaginal,
rectal, and the like. The compounds and compositions described
herein may be administered in unit dosage forms and/or formulations
containing conventional nontoxic pharmaceutically-acceptable
carriers, adjuvants, and vehicles.
[0223] Illustrative formats for oral administration include
tablets, capsules, elixirs, syrups, and the like.
[0224] Illustrative routes for parenteral administration include
intravenous, intraarterial, intraperitoneal, epidurial,
intraurethral, intrasternal, intramuscular and subcutaneous, as
well as any other art recognized route of parenteral
administration.
[0225] Depending upon the disease as described herein, the route of
administration and/or whether the compounds and/or compositions are
administered locally or systemically, a wide range of permissible
dosages are contemplated herein, including doses falling in the
range from about 1 .mu.g/kg to about 1 g/kg. The dosages may be
single or divided, and may administered according to a wide variety
of protocols, including q.d., b.i.d., t.i.d., or even every other
day, once a week, once a month, once a quarter, and the like. In
each of these cases it is understood that the therapeutically
effective amounts described herein correspond to the instance of
administration, or alternatively to the total daily, weekly, month,
or quarterly dose, as determined by the dosing protocol.
EXAMPLES
Compound Examples
[0226] The compounds described herein may be prepared using the
process and syntheses described herein, as well as using general
organic synthetic methods. In particular, methods for preparing the
compounds are described in U.S. patent application publication
2005/0002942, the disclosure of which is incorporated herein by
reference.
Example
[0227] General formation of folate-peptides. The folate-containing
peptidyl fragment Pte-Glu-(AA).sub.n-NH(CHR.sub.2)CO.sub.2H (3) is
prepared by a polymer-supported sequential approach using standard
methods, such as the Fmoc-strategy on an acid-sensitive
Fmoc-AA-Wang resin (1), as shown in the following Scheme:
##STR00053##
[0228] It is to be understood that unnatural amino acids may be
included in the foregoing process using the appropriate starting
materials.
[0229] In this illustrative embodiment of the processes described
herein, R.sub.1 is Fmoc, R.sub.2 is the desired
appropriately-protected amino acid side chain, and DIPEA is
diisopropylethylamine. Standard coupling procedures, such as PyBOP
and others described herein or known in the art are used, where the
coupling agent is illustratively applied as the activating reagent
to ensure efficient coupling. Fmoc protecting groups are removed
after each coupling step under standard conditions, such as upon
treatment with piperidine, tetrabutylammonium fluoride (TBAF), and
the like. Appropriately protected amino acid building blocks, such
as Fmoc-Glu-OtBu, Fmoc-D-Glu-OtBu, N.sup.10-TFA-Pte-OH, and the
like, are used, as described in the Scheme, and represented in step
(b) by Fmoc-AA-OH. Thus, AA refers to any amino acid starting
material, that is appropriately protected. It is to be understood
that the term amino acid as used herein is intended to refer to any
reagent having both an amine and a carboxylic acid functional group
separated by one or more carbons, and includes the naturally
occurring alpha and beta amino acids, as well as amino acid
derivatives and analogs of these amino acids. In particular, amino
acids having side chains that are protected, such as protected
serine, threonine, cysteine, aspartate, and the like may also be
used in the folate-peptide synthesis described herein. Further,
gamma, delta, or longer homologous amino acids may also be included
as starting materials in the folate-peptide synthesis described
herein. Further, amino acid analogs having homologous side chains,
or alternate branching structures, such as norleucine, isovaline,
.beta.-methyl threonine, .beta.-methyl cysteine,
.beta.,.beta.-dimethyl cysteine, and the like, may also be included
as starting materials in the folate-peptide synthesis described
herein.
[0230] The coupling sequence (steps (a) & (b)) involving
Fmoc-AA-OH is performed "n" times to prepare solid-support peptide
(2), where n is an integer and may equal 0 to about 100. Following
the last coupling step, the remaining Fmoc group is removed (step
(a)), and the peptide is sequentially coupled to a glutamate
derivative (step (c)), deprotected, and coupled to TFA-protected
pteroic acid (step (d)). Subsequently, the peptide is cleaved from
the polymeric support upon treatment with trifluoroacetic acid,
ethanedithiol, and triisopropylsilane (step (e)). These reaction
conditions result in the simultaneous removal of the t-Bu, t-Boc,
and Trt protecting groups that may form part of the
appropriately-protected amino acid side chain. The TFA protecting
group is removed upon treatment with base (step (f)) to provide the
folate-containing peptidyl fragment (3).
##STR00054##
Example
[0231] The corresponding compounds containing one or more D-amino
acids may also be prepared, such as the following:
##STR00055## ##STR00056##
and the like.
Example
[0232] Preparation of tubulysin hydrazides. Illustrated by
preparing EC0347. N,N-Diisopropylethylamine (DIPEA, 6.1 .mu.L) and
isobutyl chloroformate (3.0 .mu.L) were added with via syringe in
tandem into a solution of tubulysin B (0.15 mg) in anhydrous EtOAc
(2.0 mL) at -15.degree. C. After stirring for 45 minutes at
-15.degree. C. under argon, the reaction mixture was cooled down to
-20.degree. C. and to which was added anhydrous hydrazine (5.0
.mu.L). The reaction mixture was stirred under argon at -20.degree.
C. for 3 hours, quenched with 1.0 mM sodium phosphate buffer (pH
7.0, 1.0 mL), and injected into a preparative HPLC for
purification. Column: Waters XTerra Prep MS C.sub.18 10 .mu.m,
19.times.250 mm; Mobile phase A: 1.0 mM sodium phosphate buffer, pH
7.0; Mobile phase B: acetonitrile; Method: 10% B to 80% B over 20
minutes, flow rate=25 mL/min. Fractions from 15.14-15.54 minutes
were collected and lyophilized to produce EC0347 as a white solid
(2.7 mg). The foregoing method is equally applicable for preparing
other tubulysin hydrazides by the appropriate selection of the
tubulysin starting compound.
Example
##STR00057##
[0234] Synthesis of coupling reagent EC0311. DIPEA (0.60 mL) was
added to a suspension of
HOBt-OCO.sub.2--(CH.sub.2).sub.2--SS-2-pyridine HCl (685 mg, 91%)
in anhydrous DCM (5.0 mL) at 0.degree. C., stirred under argon for
2 minutes, and to which was added anhydrous hydrazine (0.10 mL).
The reaction mixture was stirred under argon at 0.degree. C. for 10
minutes and room temperature for an additional 30 minutes,
filtered, and the filtrate was purified by flash chromatography
(silica gel, 2% MeOH in DCM) to afford EC0311 as a clear thick oil
(371 mg), solidified upon standing.
Example
##STR00058##
[0236] Preparation of tubulysin disulfides (stepwise process).
Illustrated for EC0312. DIPEA (36 .mu.L) and isobutyl chloroformate
(13 .mu.L) were added with the help of a syringe in tandem into a
solution of tubulysin B (82 mg) in anhydrous EtOAc (2.0 mL) at
-15.degree. C. After stirring for 45 minutes at -15.degree. C.
under argon, to the reaction mixture was added a solution of EC0311
in anhydrous EtOAc (1.0 mL). The resulting solution was stirred
under argon at -15.degree. C. for 15 minutes and room temperature
for an additional 45 minutes, concentrated, and the residue was
purified by flash chromatography (silica gel, 2 to 8% MeOH in DCM)
to give EC0312 as a white solid (98 mg). The foregoing method is
equally applicable for preparing other tubulysin derivatives by the
appropriate selection of the tubulysin starting compound.
Example
##STR00059##
[0238] Hydroxydaunorubucin pyridyldisulfide. Similarly, this
compound was prepared as described herein in 65% yield, and
according to the foregoing scheme.
Example
##STR00060##
[0240] Tubulysin B pyridyldisulfide. Similarly, this compound is
prepared as described herein.
Example
##STR00061##
[0242] D-EC0488. This compound was prepared by SPPS according to
the general peptide synthesis procedure described herein starting
from H-Cys(4-methoxytrityl)-2-chlorotrityl-Resin, and the following
SPPS reagents:
TABLE-US-00002 Reagents mmol equivalent MW amount
H-D-Cys(4-methoxytrityl)-2- 0.10 0.17 g chlorotrityl-Resin (loading
0.6 mmol/g) D-EC0475 0.13 1.3 612.67 0.082 g Fmoc-D-Glu(OtBu)-OH
0.19 1.9 425.47 0.080 g D-EC0475 0.13 1.3 612.67 0.082 g
Fmoc-D-Glu(OtBu)-OH 0.19 1.9 425.47 0.080 g D-EC0475 0.13 1.3
612.67 0.082 g Fmoc-D-Glu-OtBu 0.19 1.9 425.47 0.080 g
N.sup.10TFA-Pteroic Acid 0.16 1.6 408.29 0.066 g (dissolve in 10 ml
DMSO) DIPEA 2.0 eq of AA PyBOP 1.0 eq of AA
[0243] Coupling steps. In a peptide synthesis vessel add the resin,
add the amino acid solution, DIPEA, and PyBOP. Bubble argon for 1
hr. and wash 3.times. with DMF and IPA. Use 20% piperidine in DMF
for Fmoc deprotection, 3.times.(10 min), before each amino acid
coupling. Continue to complete all 9 coupling steps. At the end
treat the resin with 2% hydrazine in DMF 3.times.(5 min) to cleave
TFA protecting group on Pteroic acid, wash the resin with DMF
(3.times.), IPA (3.times.), MeOH (3.times.), and bubble the resin
with argon for 30 min.
[0244] Cleavage step. Reagent: 92.5% TFA, 2.5% H.sub.2O, 2.5%
triisopropylsilane, 2.5% ethanedithiol. Treat the resin with
cleavage reagent 3.times.(10 min, 5 min, 5 min) with argon
bubbling, drain, wash the resin once with cleavage reagent, and
combine the solution. Rotavap until 5 ml remains and precipitate in
diethyl ether (35 mL). Centrifuge, wash with diethyl ether, and
dry. About half of the crude solid (-100 mg) was purified by
HPLC.
[0245] HPLC Purification step. Column: Waters Xterra Prep MS C18 10
.mu.m 19.times.250 mm; Solvent A: 10 mM ammonium acetate, pH 5;
Solvent B: ACN; Method: 5 min 0% B to 25 min 20% B 26 mL/min.
Fractions containing the product was collected and freeze-dried to
give 43 mg EC0488 (51% yield). .sup.1H NMR and LC/MS (exact mass
1678.62) were consistent with the product.
Example
##STR00062##
[0247] General Synthesis of Disulfide Containing Tubulysin
Conjugates. Illustrated with pyridinyl disulfide derivatives of
certain naturally occurring tubulysins, where R.sup.1 is H or OH,
and R.sup.10, is alkyl or alkenyl. A binding ligand-linker
intermediate containing a thiol group is taken in deionized water
(ca. 20 mg/mL, bubbled with argon for 10 minutes prior to use) and
the pH of the suspension was adjusted by saturated NaHCO.sub.3
(bubbled with argon for 10 minutes prior to use) to about 6.9 (the
suspension may become a solution when the pH increased). Additional
deionized water is added (ca. 20-25%) to the solution as needed,
and to the aqueous solution is added immediately a solution of
EC0312 in THF (ca. 20 mg/mL). The reaction mixture becomes
homogenous quickly. After stirring under argon, e.g. for 45
minutes, the reaction mixture is diluted with 2.0 mM sodium
phosphate buffer (pH 7.0, ca 150 volume percent) and the THF is
removed by evacuation. The resulting suspension is filtered and the
filtrate may be purified by preparative HPLC (as described herein).
Fraction are lyophilized to isolate the conjugates. The foregoing
method is equally applicable for preparing other tubulysin
conjugates by the appropriate selection of the tubulysin starting
compound.
Example
##STR00063##
[0249] General Method 2 for Preparing Conjugates (one-pot).
Illustrated with preparation of EC1456. DIPEA (7.8 .mu.L) and
isobutyl chloroformate (3.1 .mu.L) were added with the help of a
syringe in tandem into a solution of tubulysin A (18 mg) in
anhydrous EtOAc (0.50 mL) at -15.degree. C. After stirring for 35
minutes at -15.degree. C. under argon, to the reaction mixture was
added a solution of EC0311 (5.8 mg) in anhydrous EtOAc (0.50 mL).
The cooling was removed and the reaction mixture was stirred under
argon for an additional 45 minutes, concentrated, vacuumed, and the
residue was dissolved in THF (2.0 mL). Meanwhile, D-EC0488 (40 mg)
was dissolved in deionized water (bubbled with argon for 10 minutes
prior to use) and the pH of the aqueous solution was adjusted to
6.9 by saturated NaHCO.sub.3. Additional deionized water was added
to the D-EC0488 solution to make a total volume of 2.0 mL and to
which was added immediately the THF solution containing the
activated tubulysin. The reaction mixture, which became homogeneous
quickly, was stirred under argon for 50 minutes and quenched with
2.0 mM sodium phosphate buffer (pH 7.0, 15 mL). The resulting
cloudy solution was filtered and the filtrate was injected into a
preparative HPLC for purification. Column: Waters XTerra Prep MS
C.sub.18 10 .mu.m, 19.times.250 mm; Mobile phase A: 2.0 mM sodium
phosphate buffer, pH 7.0; Mobile phase B: acetonitrile; Method: 1%
B for 5 minutes, then 1% B to 60% B over the next 30 minutes, flow
rate=26 mL/min. Fractions from 20.75-24.50 minutes were collected
and lyophilized to afford EC1456 as a pale yellow fluffy solid (26
mg). The foregoing method is equally applicable for preparing other
tubulysin and other conjugates by the appropriate selection of the
tubulysin or other drug starting compound.
Example
[0250] EC1426 is prepared according to the following process.
##STR00064## ##STR00065##
Example
[0251] EC1456 is prepared according to the following process.
##STR00066## ##STR00067##
Example
[0252] N.sup.10-TFA Protected EC1454 is prepared according to the
following process.
##STR00068##
Example
[0253] EC1454 is prepared according to the following process.
##STR00069##
Example
[0254] EC1004 is prepared according to the following process.
##STR00070##
[0255] Into a round bottomed flask equipped with magnetic stir bar
and temperature probe dipeptide EC1458, imidazole, and methylene
chloride is added. Once all the solids have dissolved, the solution
is cooled using an ice bath. Chlorotriethylsilane (TESC1) is added
drop wise and the ice bath is removed. The reaction is monitored
for completion. A second portion of chlorotriethylsilane and/or
imidazole is added if necessary. The imidazole HCl salt is removed
by filtration and methylene chloride is added. The organics are
washed with a saturated solution of sodium chloride (brine), the
aqueous layer is back extracted once with methylene chloride, and
the combined organic layers are washed with brine. The organic
layer is dried over sodium sulfate and concentrated on a rotary
evaporator. The residue is dissolved in tetrahydrofuran (THF) and
cooled to approximately -45.degree. C. A solution of potassium
bis(trimethylsilyl)amide (KHMDS) in toluene is added drop wise.
With stirring, chloromethyl butyrate is added and the reaction is
monitored. The reaction is quenched with methanol and then ethyl
acetate and brine are added. The aqueous layer is discarded and the
organics are washed once with brine. The organic layer is
concentrated on a rotary evaporator and the oily residue is passed
through a short plug of silica gel. The plug is washed with a 20%
solution of ethyl acetate in petroleum ether. The combined organics
are concentrated on a rotary evaporator until distillation ceases.
The crude EC1004 oil is analyzed by LC and NMR and stored in a
freezer until use.
Example
[0256] EC1005 is prepared according to the following process.
##STR00071##
[0257] Into an appropriately sized hydrogenation flask place
R--N-methyl pipecolinate (MEP), pentafluorophenol,
N-methylpyrrolidinone (NMP), and ethyl dimethylaminopropyl
carbodiimide (EDC). The mixture is stirred for at least 16 h.
EC1004 dissolved in N-methyl pyrrolidinone (NMP) and 10 wt % Pd/C
are added. The reaction mixture is stirred/shaken under hydrogen
pressure until the reaction is complete by LC analysis. The Pd/C is
removed by filtration through celite. The celite is washed with
ethyl acetate and the combined organics are washed three times with
a 1% sodium bicarbonate/10% sodium chloride solution. The organic
layer is dried over sodium sulfate and concentrated on a rotary
evaporator. The residue is dissolved in DCM and purified by silica
gel chromatography using ethyl acetate and petroleum ether as
eluents. Fractions are collected, checked for purity, combined and
dried on a rotary evaporator. The EC1005 oil is assayed by LC and
stored in a freezer until use.
Example
[0258] EC1008 is prepared according to the following process.
##STR00072##
[0259] EC1005 is dissolved in 1,2-dichloroethane (DCE) and
trimethyltin hydroxide is added. The reaction mixture is heated and
reaction is monitored by LC. On completion, the mixture is cooled
with an ice bath and filtered. The solids are then washed with DCE.
The organic layer is washed once with water and dried over sodium
sulfate. The solution is concentrated on a rotary evaporator and
the residue dissolved in tetrahydrofuran (THF). Triethylamine
trihydrofluoride is added and the mixture stirred while monitoring
with LC. Pyridine, dimethylaminopyridine (DMAP), and acetic
anhydride are added. The reaction is stirred and monitored by LC.
The reaction mixture is concentrated to a residue and the product
is purified by C18 column chromatography with acetonitrile and
water as eluents. Product fractions are collected, concentrated,
and lyophilized to yield a white to off-white powder.
Example
[0260] EC1426 is prepared according to the following process.
##STR00073##
[0261] EC1422 is dissolved in tetrahydrofuran (THF) and
(Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate
(PyB op) and diisopropylethylamine (DIPEA) are added. Once all the
solids have dissolved hydrazine is added and the reaction is
stirred and monitored for completion. EC0607 is added and the
mixture stirred and monitored for completion by LC. Ethyl acetate
is added and the organics are washed once with saturated ammonium
chloride, twice with saturated sodium bicarbonate, and once with
saturated sodium chloride. The organics are dried over sodium
sulfate and concentrated on a rotary evaporator. The crude EC1426
is purified by silica column chromatography with dichloromethane
and methanol as eluents. Fractions are collected and the combined
product fractions are concentrated on a rotary evaporator to yield
a yellow solid.
Example
[0262] EC1428 is prepared according to the following process.
##STR00074##
[0263] EC1008 is dissolved in dichloromethane and pentafluorophenol
dissolved in DCM along with N-cyclohexylcarbodiimide,N'-methyl
polystyrene (DCC-resin) are added. The mixture is stirred and
reaction completion is monitored by LC. The mixture is filtered to
remove the resin and the organic layer is concentrated on a rotary
evaporator to yield activated EC1008. In a separate flask, EC1426
is dissolved in dichloromethane and trifluoroacetic acid is added.
The reaction mixture is stirred and monitored for completion by LC.
The reaction mixture is concentrated on a rotary evaporator to
yield deprotected EC1426. The activated EC1008 is dissolved in DMF
and diisopropylethylamine (DIPEA) is added. The deprotected EC1426
is dissolved in DMF and added to the reaction mixture. The reaction
is stirred and monitored for completion by LC. Ethyl acetate is
added and the organics are washed three times with saturated
aqueous sodium chloride. The organic layer is dried over sodium
sulfate and the volatiles removed by rotary evaporation. The crude
EC1428 is purified by silica column chromatography using
dichloromethane and methanol as eluents. Fractions are collected,
checked for purity, and the combined product fractions are
concentrated by rotary evaporation to yield a yellow solid. The
EC1428 is stored in a freezer.
Example
[0264] EC1454 is prepared according to the following process.
##STR00075##
[0265] The solid phase synthesis of N.sup.10-TFA protected EC1454
starts with resin bound trityl protected D-cysteine. The resin is
suspended in dimethylformamide (DMF) and washed twice with DMF.
EC0475 (glucamine modified L-glutamic acid),
(Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate
(PyBOP), and diisopropylethylamine (DIPEA) are added to reaction
mixture. After at least 1 hour, a Kaiser test is performed to
ensure the coupling is complete. The resin is washed three times
with DMF, three times with IPA, and three times with DMF. The resin
is slowly washed three times with piperidine in DMF, three times
with DMF, and three times with IPA. A Kaiser test is performed to
confirm deprotection. The resin is washed three times with DMF and
the next amino acid in the sequence is coupled following the same
process. Monomers are coupled in the following order: 1) EC0475, 2)
Fmoc-D-Glu(OtBu)-OH, 3) EC0475, 4) Fmoc-D-Glu(OtBu)-OH, 5) EC0475,
6) Fmoc-D-Glu-OtBu, and 7) N.sup.10-TFA-Pte-OH.
[0266] Once the final coupling is complete, the resin is washed
three times with methanol and dried by passing argon through the
resin at room temperature. The dried resin is suspended in a
mixture of TFA, water, ethanedithiol, and triisopropylsilane. After
1 hour the resin is removed by filtration and washed with TFA. The
product is precipitated by addition to cold ethyl ether, filtered,
and washed with ether. The solids are dried under vacuum at room
temperature and stored in a freezer.
##STR00076##
[0267] N.sup.10-TFA EC1454 is dissolved in argon sparged water.
Sodium carbonate (1M in water, argon sparged) is added to achieve a
pH of 9.4-10.1. The reaction mixture is stirred for at least 20
minutes. Once the reaction is complete as determined by LC, it is
quenched by adjusting the pH to 1.9-2.3 with 2M HCl. The product is
purified by C18 column chromatography using acetonitrile and pH 5
ammonium acetate buffer as eluents. Fractions are collected and
checked for purity by HPLC. The combined product fractions are
concentrated on a rotary evaporator and then lyophilized to yield
EC1454 as a yellow solid. The product is stored at -20.degree.
C.
Example
[0268] EC1456 is prepared according to the following process.
##STR00077##
[0269] EC1428 is dissolved in acetonitrile and a solution of EC1454
in pH 7.4 Sodium phosphate buffer is added. The solutions are
sparged with argon before and after addition. The reaction mixture
is stirred for at least 15 minutes and then checked for completion.
The desired product is purified by C18 column chromatography using
acetonitrile and pH 7.4 phosphate buffer as eluents. The product
fractions are collected, checked for purity, combined and
concentrated by ultra-filtration to yield an aqueous solution that
is 10-20 mg/mL EC1456. The final product solution is sampled for
assay and then stored in a freezer.
[0270] The positive electrospray mass spectrum of EC1456 was
obtained on a high resolution Waters Acquity UPLC Xevo Gs-S QTOF
mass spectrometer. The spectrum was obtained following separation
of the major component on a UPLC inlet system, the resolving power
was approximately 35,000. The accurate mass measurement of the M+H
monoisotopic peak was 2625.0598, which is 1.1 ppm error difference
from the theoretical value of 2625.0570 for an ion of formula
C.sub.110H.sub.166N.sub.23O.sub.45S.sub.3. The isotopic
distribution is also consistent with that formula.
[0271] Mass spectral features of the ES+ spectrum for EC1456
TABLE-US-00003 Observed Ion Interpretation 2626.06 .sup.13C isotope
of the (M + H).sup.+ ion for the MW 2624 drug substance 1313.54
.sup.13C isotope of the (M + 2H).sup.++ ion for the MW 2624 drug
substance 1150.43 .sup.13C isotope of the (M + 2H - 326).sup.++
fragment, corresponding to the cleavage of the peptide bond at the
tertiary nitrogen and the loss of the butyric acid moiety. 876.03
.sup.13C isotope of the (M + 3H).sup.+++ ion for the MW 2624 drug
substance 657.27 .sup.13C isotope of the (M + 4H).sup.++++ ion for
the MW 2624 drug substance
[0272] A sample of .about.30 mg EC1456 was dissolved in 665 .mu.L
of a 9:1 mixture of deuterated dimethylsulfoxide and deuterated
water. The .sup.1H NMR spectrum was obtained at 500 MHz at 26 deg.
C. on an Agilent model DD2 spectrometer fitted with a 2 channel
probe containing both broadband and proton observe coils. The
.sup.13C NMR spectrum was obtained at 125 MHz on the same
instrument under identical conditions. All spectra were referenced
to the DMSO solvent residual signals at 2.5 ppm (.sup.1H) and 39.50
ppm (.sup.13C).
[0273] All spectral features are assigned for both NMR spectra in
the tables below (.sup.1H and .sup.13C) using the atom numbering in
the following figure (where the * symbols indicates the connection
for the disulfide bond).
##STR00078##
Assignments were made on the basis of both 1D and 2D NMR
experiments, including through bond H--H connectivity using the
COSY and TCSY 2D experiments, through space H--H proximity using 2D
NOESY, carbon multiplicity measurement using the 1D DEPT experiment
and through bond C--H connectivity using the proton detected 2D
experiments HSQC and HMBC. In most cases of overlap in the 1D
spectra (different protons or carbons resonating at the same
chemical shift) could be resolved in the 2D spectra, in these cases
the tables reflect the chemical shifts measured from the 2D spectra
but summed integrations for the group of co-resonating species. In
some cases of 1D overlap (such as the nearly identical glutamic
acid and glucamine subunits) there was also overlap in the 2D
correlation spectra which precludes unambiguous assignment of
single or multiple resonances between multiple atom numbers, in
these cases there are multiple entries for chemical shift and/or
atom number assignments in a single table row.
[0274] NH and OH protons were exchanged by the D.sub.2O deuterium
atoms and are mostly absent from the spectrum, except weak broad
peaks in the 5-10 ppm region. The .sup.1H peaks in the spectrum
that are not listed in the table include a broad HOD peak at 3.75
ppm, and a DMSO peak at 2.50 ppm. The HOD peak does not obscure any
resonances, but elevates the integrations for nearby resonances at
4.2 and 3.4-3.7 ppm due to the broad baseline rise. The DMSO peak
obscures the resonance for H129, which is not integrated for this
reason. The .sup.13C peaks in spectrum not listed in the table
include the very large DMSO solvent at 39.50 ppm. The DMSO peak
obscures both the signals from C91 and C93. The C116 peak is not
observable in the .sup.13C spectrum due to extensive broadening due
to conformational changes around the nearby amide group. All three
chemical shifts (C91, C93, C116) are visible in and measured in the
proton detected 2D correlation spectra.
Proton NMR assignments for EC1456
TABLE-US-00004 Proton Chemical Shift (ppm) Assignment # protons
8.61 5 1 8.16 103 1 7.58 15, 17 2 6.96 95, 99 2 6.62 14, 18 4 6.59
96, 98 6.18 116 Ha 1 5.7 107 1 5.24 116 Hb 1 4.47 11 2 4.39 111,
122 2 4.21 78 10 4.21 65 4.18 84 4.15 46 4.15 59 4.13 21 4.13 40
4.09 27 4.09 92 3.61 33, 52, 71 3 3.56 34, 53, 72 6 3.54 37Ha,
56Ha, 75Ha 3.46 36, 55, 74 3 3.4 35, 54, 73 6 3.38 37Hb, 56Hb, 75Hb
3.21 80Ha, 32Ha, 51 Ha, 4 70 Ha 3.05 32Hb, 51Hb, 70Hb 3 2.93 80 Hb
3 2.91 83 2.8 133Ha 1 2.68 93 2 2.49 (see text) 129 1 2.35 89 2
2.33 110Ha 2.8 133Hb 37 2.17 118 2.14-2.08 24, 29, 42, 48, 61, 67
2.09 110Hb 2.08 109 2.02 135 1.97-1.70 28, 41, 47, 60, 66 1.92 23Ha
1.88 123 1.8 91Ha 1.79 23Hb 1.77 112 1.6 131Ha 9 1.56 130Ha 1.5
132Ha 1.5 91Hb 1.45 125Ha 1.42 119 1.4 132Hb 1.33 130Hb 1.14 131Hb
2 1.07 125Hb 1 90 3 0.94 114 3 0.79 124 3 0.77 126 3 0.75 120 3
0.64 113 3
Carbon NMR assignments for EC1456
TABLE-US-00005 Carbon Chemical shift (ppm) Assignment 176.77,
176.32 43, 62 175.74 88 175.42 22 174.75 121 173.87, 172.68, 25,
38, 44, 57, 63 172.15, 171.94, 171.84 173.43 79 173.3 128 172.79
(2x), 172.72 30, 49, 68 172.46 117 170.87 76 170.39 108 169.3 105
166.09 19 162.4 9 160.7 101 156.4 85 156.09 3 155.71 97 154.59 1
150.84 13 149.63 102 149.11 6 148.99 5 130.44 95, 99 128.99 15, 17
128.89 94 127.99 8 124.97 103 122.24 16 115.25 96, 98 111.86 14, 18
72.17 (3x) 35, 54, 73 71.78, 71.74, 71.71 33, 52, 71 71.62, 71.59
(2x) 36, 55, 74 69.65, 69.57 (2x) 34, 53, 72 69.45 107 69.34 116
68.51 129 63.42 (3x) 37, 56, 75 63.03 84 55.08 133 54.05 40 53.88
78 53.46 (2x) 46, 59 53.33 27 52.96 (2x) 122, 111 52.89 21 52.55 65
49.77 92 46.07 11 44.02 135 42.85 80 42.34 (2x), 42.29 32, 51, 70
39.52 93 38.95 91 37.43 83 35.95 118 35.43 123 35.38 89 34.86 110
32.56, 32.36, 24, 29, 42, 48, 61, 67 32.16, 32.09 (2x), 31.81 30.5
112 29.95 130 28.60, 28.04, 27.78 28, 41, 47, 60, 66 (2x), 27.66 27
23 25.01 132 24.43 125 23.04 131 20.86 109 20.56 114 19.64 113
18.36 90 18.04 119 15.64 124 13.72 120 10.28 126
[0275] The IR spectrum of EC1456 was acquired on a Nexus 6700.RTM.
Fourier transform infrared (FT-IR) spectrophotometer (Thermo
Nicolet) equipped with an Ever-Glo mid/far IR source, an extended
range potassium bromide (KBr) beam splitter, and a deuterated
triglycine sulfate (DTGS) detector. An attenuated total reflectance
(ATR) accessory (Thunderdome.TM., Thermo Spectra-Tech), with a
germanium (Ge) crystal was used for data acquisition. The spectrum
represents 256 co-added scans collected at a spectral resolution of
4 cm.sup.-1. A background data set was acquired with a clean Ge
crystal. A Log 1/R (R=reflectance) spectrum was acquired by taking
a ratio of these two data sets against each other. Wavelength
calibration was performed using polystyrene.
Infrared band assignments for EC1456 reference substance
TABLE-US-00006 Characteristic Absorption(s) (cm.sup.-1) Functional
Group 1700-1500 (m, m) Aromatic C.dbd.C Bending 2950-2850 (m or s)
Alkyl C--H Stretch ~3030 (v) Aromatic C--H Stretch 3550-3200
(broad, s) Alcohol/Phenol O--H Stretch 3700-3500 (m) Amide C.dbd.O
Stretch
[0276] The ultraviolet spectrum EC 1456 acquired on a Perkin-Elmer
Lambda 25 UV/Vis spectrometer. The spectrum was recorded at 40.7 uM
in 0.1M NaOH solvent on a 1 cm path-length cell at 25 deg. C. The
local maxima at 366 nm, 288 nm and 243 nm are due primarily to the
Pteroic acid, benzamide/phenol and thiazole-amide substructures,
respectively, although the molecule contains dozens of chromaphores
with overlapping absorption in the UV region.
Example
[0277] The following additional compounds are described and are
prepared according to the general processes described herein.
##STR00079## ##STR00080## ##STR00081## ##STR00082## ##STR00083##
##STR00084## ##STR00085##
Method Examples
[0278] General. The following abbreviations are used herein:
partial response (PR); complete response (CR), three times per week
(M/W/F) (TIW).
[0279] Method.
[0280] Relative Affinity Assay. The affinity for folate receptors
(FRs) relative to folate was determined according to a previously
described method (Westerhof, G. R., J. H. Schornagel, et al. (1995)
Mol. Pharm. 48: 459-471) with slight modification. Briefly,
FR-positive KB cells were heavily seeded into 24-well cell culture
plates and allowed to adhere to the plastic for 18 h. Spent
incubation media was replaced in designated wells with folate-free
RPMI (FFRPMI) supplemented with 100 nM .sup.3H-folic acid in the
absence and presence of increasing concentrations of test article
or folic acid. Cells were incubated for 60 min at 37.degree. C. and
then rinsed 3 times with PBS, pH 7.4. Five hundred microliters of
1% SDS in PBS, pH 7.4, were added per well. Cell lysates were then
collected and added to individual vials containing 5 mL of
scintillation cocktail, and then counted for radioactivity.
Negative control tubes contained only the .sup.3H-folic acid in
FFRPMI (no competitor). Positive control tubes contained a final
concentration of 1 mM folic acid, and CPMs measured in these
samples (representing non-specific binding of label) were
subtracted from all samples. Notably, relative affinities were
defined as the inverse molar ratio of compound required to displace
50% of .sup.3H-folic acid bound to the FR on KB cells, and the
relative affinity of folic acid for the FR was set to 1.
[0281] Method.
[0282] Inhibition of Cellular DNA Synthesis. The compounds
described herein were evaluated using an in vitro cytotoxicity
assay that predicts the ability of the drug to inhibit the growth
of folate receptor-positive KB cells. The compounds were comprised
of folate linked to a respective chemotherapeutic drug, as prepared
according to the protocols described herein. The KB cells were
exposed for up to 7 h at 37.degree. C. to the indicated
concentrations of folate-drug conjugate in the absence or presence
of at least a 100-fold excess of folic acid. The cells were then
rinsed once with fresh culture medium and incubated in fresh
culture medium for 72 hours at 37.degree. C. Cell viability was
assessed using a .sup.3H-thymidine incorporation assay. For
compounds described herein, dose-dependent cytotoxicity was
generally measurable, and in most cases, the IC.sub.50 values
(concentration of drug conjugate required to reduce
.sup.3H-thymidine incorporation into newly synthesized DNA by 50%)
were in the low nanomolar range. Furthermore, the cytotoxicities of
the conjugates were reduced in the presence of excess free folic
acid, indicating that the observed cell killing was mediated by
binding to the folate receptor.
[0283] Method.
[0284] In vitro test against the various cancer cell lines. IC50
values were generated for various cell lines and the results are
shown in the table below. Cells are heavily seeded in 24-well
Falcon plates and allowed to form nearly confluent monolayers
overnight. Thirty minutes prior to the addition of the test
compound, spent medium is aspirated from all wells and replaced
with fresh folate-deficient RPMI medium (FFRPMI). A subset of wells
are designated to receive media containing 100 .mu.M folic acid.
The cells in the designated wells are used to determine the
targeting specificity. Without being bound by theory it is
suggested that the cytotoxic activity produced by test compounds in
the presence of excess folic acid, i.e. where there is competition
for FR binding, corresponds to the portion of the total activity
that is unrelated to FR-specific delivery. Following one rinse with
1 mL of fresh FFRPMI containing 10% heat-inactivated fetal calf
serum, each well receives 1 mL of medium containing increasing
concentrations of test compound (4 wells per sample) in the
presence or absence of 100 .mu.M free folic acid as indicated.
Treated cells are pulsed for 2 h at 37.degree. C., rinsed 4 times
with 0.5 mL of media, and then chased in 1 mL of fresh medium up to
70 h. Spent medium is aspirated from all wells and replaced with
fresh medium containing 5 .mu.Ci/mL .sup.3H-thymidine. Following a
further 2 h 37.degree. C. incubation, cells are washed 3 times with
0.5 mL of PBS and then treated with 0.5 mL of ice-cold 5%
trichloroacetic acid per well. After 15 min, the trichloroacetic
acid is aspirated and the cell material solubilized by the addition
of 0.5 mL of 0.25 N sodium hydroxide for 15 min. A 450 .mu.L
aliquot of each solubilized sample is transferred to a
scintillation vial containing 3 mL of Ecolume scintillation
cocktail and then counted in a liquid scintillation counter. Final
tabulated results are expressed as the percentage of
.sup.3H-thymidine incorporation relative to untreated controls.
[0285] Method.
[0286] Human serum stability. Compounds described herein are tested
in human serum for stability using conventional protocols and
methods.
[0287] Method.
[0288] Inhibition of Tumor Growth in Mice. Four to seven week-old
mice (Balb/c or nu/nu strains) were purchased from Harlan Sprague
Dawley, Inc. (Indianapolis, Ind.). Normal rodent chow contains a
high concentration of folic acid (6 mg/kg chow); accordingly, mice
used were maintained on the folate-free diet (Harlan diet #TD00434)
for 1 week before tumor implantation to achieve serum folate
concentrations close to the range of normal human serum. For tumor
cell inoculation, 1.times.10.sup.6 M109 cells (Balb/c strain) or
1.times.10.sup.6 KB cells (nu/nu strain) in 100 .mu.L were injected
in the subcutis of the dorsal medial area. Tumors were measured in
two perpendicular directions every 2-3 days using a caliper, and
their volumes were calculated as 0.5.times.L.times.W.sup.2, where
L=measurement of longest axis in mm and W=measurement of axis
perpendicular to L in mm. Log cell kill (LCK) and treated over
control (T/C) values were then calculated according to published
procedures (see, e.g., Lee et al., "BMS-247550: a novel epothilone
analog with a mode of action similar to paclitaxel but possessing
superior antitumor efficacy" Clin Cancer Res 7:1429-1437 (2001);
Rose, "Taxol-based combination chemotherapy and other in vivo
preclinical antitumor studies" J Natl Cancer Inst Monogr 47-53
(1993)). Dosing solutions were prepared fresh each day in PBS and
administered through the lateral tail vein of the mice. Dosing was
initiated when the s.c. tumors had an average volume between 50-100
mm.sup.3 (t.sub.0), typically 8 days post tumor inoculation (PTI)
for KB tumors, and 11 days PTI for M109 tumors.
[0289] Method.
[0290] Drug Toxicity determinations. Persistent drug toxicity was
assessed by collecting blood via cardiac puncture and submitting
the serum for independent analysis of blood urea nitrogen (BUN),
creatinine, total protein, AST-SGOT, ALT-SGPT plus a standard
hematological cell panel at Ani-Lytics, Inc. (Gaithersburg, Md.).
In addition, histopathologic evaluation of formalin-fixed heart,
lungs, liver, spleen, kidney, intestine, skeletal muscle and bone
(tibia/fibula) were conducted by board-certified pathologists at
Animal Reference Pathology Laboratories (ARUP; Salt Lake City,
Utah).
[0291] Method.
[0292] General KB Tumor Assay. The anti-tumor activity of the
compounds described herein, when administered intravenously (i.v.)
to tumor-bearing animals, was evaluated in nu/nu mice bearing
subcutaneous KB tumors. Approximately 8 days post tumor inoculation
in the subcutis of the right axilla with 1.times.10.sup.6 KB cells
(average tumor volume at t.sub.o=50-100 mm.sup.3), in mice
(5/group) were injected i.v. three times a week (TIW), for 3 weeks
with 5 mmol/kg of the drug delivery conjugate or with an equivalent
dose volume of PBS (control), unless otherwise indicated. Tumor
growth was measured using calipers at 2-day or 3-day intervals in
each treatment group. Tumor volumes were calculated using the
equation V=a.times.b.sup.2/2, where "a" is the length of the tumor
and "b" is the width expressed in millimeters.
[0293] Method.
[0294] General M109 Tumors Assay. The anti-tumor activity of the
compounds described herein, when administered intravenously (i.v.)
to tumor-bearing animals, was evaluated in Balb/c mice bearing
subcutaneous M109 tumors (a syngeneic lung carcinoma).
Approximately 11 days post tumor inoculation in the subcutis of the
right axilla with 1.times.10.sup.6 M109 cells (average tumor volume
at t.sub.o=60 mm.sup.3), mice (5/group) were injected i.v. three
times a week (TIW), for 3 weeks with 1500 nmol/kg of the drug
delivery conjugate or with an equivalent dose volume of PBS
(control). Tumor growth was measured using calipers at 2-day or
3-day intervals in each treatment group. Tumor volumes were
calculated using the equation V=a.times.b.sup.2/2, where "a" is the
length of the tumor and "b" is the width expressed in
millimeters.
[0295] Method.
[0296] General 4T-1 Tumor Assay. Six to seven week-old mice (female
Balb/c strain) were obtained from Harlan, Inc., Indianapolis, Ind.
The mice were maintained on Harlan's folate-free chow for a total
of three weeks prior to the onset of and during this experiment.
Folate receptor-negative 4T-1 tumor cells (1.times.10.sup.6 cells
per animal) were inoculated in the subcutis of the right axilla.
Approximately 5 days post tumor inoculation when the 4T-1 tumor
average volume was .about.100 mm.sup.3, mice (5/group) were
injected i.v. three times a week (TIW), for 3 weeks with 3
.mu.mol/kg of drug delivery conjugate or with an equivalent dose
volume of PBS (control), unless otherwise indicated herein. Tumor
growth was measured using calipers at 2-day or 3-day intervals in
each treatment group. Tumor volumes were calculated using the
equation V=a.times.b.sup.2/2, where "a" is the length of the tumor
and "b" is the width expressed in millimeters.
[0297] Method.
[0298] Toxicity as Measured by Weight Loss. The percentage weight
change of the mice was determined in mice (5 mice/group) on
selected days post-tumor inoculation (PTI), and graphed.
[0299] Method.
[0300] Adjuvant-Induced Arthritis (AIA) Model. Female Lewis rats
were fed a folate-deficient diet (Harlan Teklad, Indianapolis,
Ind.) for 9-10 days prior to arthritis induction. The
adjuvant-induced arthritis (AIA) was induced by intradermal
inoculation (at the base of tail) of 0.5 mg of heat-killed
Mycobacteria butyricum (BD Diagnostic Systems, Sparks, Md.) in 100
.mu.L light mineral oil (Sigma). Ten days after arthritis
induction, paw edema in rats was assessed using a modified
arthritis scoring system: 0=no arthritis; 1=swelling in one type of
joint; 2=swelling in two types of joint; 3=swelling in three types
of joint; 4=swelling of the entire paw. A total score for each rat
is calculated by summarizing the scores for each of the four paws,
giving a maximum score of 16 for each rat. On Day 10 post arthritis
induction, rats with a total arthritis score of .gtoreq.2 were
removed from the study and the remaining rats were distributed
evenly across the control and treatment groups (n=5 for all groups
except that n=2-3 for healthy controls). All treatments started on
Day 10 unless mentioned otherwise.
[0301] Method.
[0302] Collagen-Induced Arthritis (CIA) Model. The collagen-induced
arthritis (CIA) was induced in female Lewis rats on
folate-deficient diet (Harlan Teklad, Indianapolis, Ind.). On Day
0, rats were immunized with 500 .mu.g of bovine collagen Type II
(Chondrex, Redmond, Wash.) formulated with Freund's complete
adjuvant. A booster immunization was given on Day 7 with 250 .mu.g
of the bovine collagen formulated with Freund's incomplete
adjuvant. Arthritis disease was assessed by a qualitative clinical
score system described by the manufacturer (Chondrex, Redmond,
Wash.): 0=normal, 1=Mild, but definite redness and swelling of the
ankle or wrist, or apparent redness and swelling limited to
individual digits, regardless of the number of affected digits,
2=Moderate redness and swelling of ankle of wrist, 3=Severe redness
and swelling of the entire paw including digits, and 4=Maximally
inflamed limb with involvement of multiple joints. On Day 10 post
first immunization, rats were distributed evenly (according to the
arthritis score) across the control and treatment groups. The CIA
rats were given ten consecutive subcutaneous doses of EC0746 and
methotrexate on days 10-19. For both drugs, an induction dose (500
nmol/kg) was given on days 10 and 15 and a maintenance dose (100
nmol/kg) was given on days 11-14 and 16-19. The animals in the
arthritis control group were left untreated. The arthritis score
and animal body weight were recorded five times a week. The result
showed that EC0746 was also effective in rats with collagen-induced
arthritis. See FIG. 13, Panels A and B.
[0303] Method.
[0304] Animal Experimental Autoimmune Uveitis Model. Experimental
autoimmune uveitis (EAU) was induced in female Lewis rats
maintained on a folate-deficient diet (Harlan Teklad, Indianapolis,
Ind.). On Day 0, the animals were immunized subcutaneously with 25
.mu.g of bovine S-Ag PDSAg peptide formulated with Freund's
incomplete adjuvant containing 0.5 mg of M. Tuberculosis H37Ra.
Purified pertussis toxin (PT) was given at a dosage of 1 .mu.g per
animal on the same day via intraperitoneal injection. The severity
of uveitis in each eye was assessed by a qualitative visual score
system: 0=No disease, eye is translucent and reflects light (red
reflex); 0.5 (trace)=Dilated blood vessels in the iris, 1=Engorged
blood vessels in iris, abnormal pupil contraction; 2=Hazy anterior
chamber, decreased red reflex; 3=Moderately opaque anterior
chamber, but pupil still visible, dull red reflex; and 4=Opaque
anterior chamber and obscured pupil, red reflex absent, proptosis.
This assessment yields a maximum uveitis score of 8 per animal.
FIG. 34 shows images the eyes of an animal (upper right) with
severe uveitis on its right eye (bottom) and a healthy eye (upper
right).
[0305] Method.
[0306] In vivo activity against tumors. Compounds described herein
show high potency and efficacy against KB tumors in nu/nu mice.
Compounds described herein show specific activity against folate
receptor expressing tumors, with low host animal toxicity. For
example, EC1456 shows a complete response in 4/4 test animals when
administered intravenously at 1 .mu.mol/kg TIW, 2 wk. EC1456 also
shows specific activity mediated by the folate receptor as
evidenced being competable with excess EC0923 (50 or 100
.mu.mol/kg), as shown in FIG. 1A. EC1456 does not show any evidence
of whole animal toxicity, as shown in FIG. 1B.
[0307] Method.
[0308] Triple negative breast cancer (TNBC) is a subtype
characterized by lack of gene expression for estrogen, progesterone
and Her2/neu. TNBC is difficult to treat, and its death rate is
disproportionately higher than for any other subtype of breast
cancer. When tested against an established triple negative
FR-positive subcutaneous MDA-MB-231 breast cancer xenograft, EC1456
was found to be highly active at 2 .mu.mmol/kg intravenous dose
administered on a three times per week, 2 consecutive week schedule
produced 4 of 5 cures, as shown in FIG. 2A. The anti-tumor activity
was not accompanied by significant weight loss in the test animals,
as shown in FIG. 2B.
[0309] Method.
[0310] A human cisplatin-resistant cell line was created by
culturing FR-positive KB cells in the presence of increasing
cisplatin concentrations (100.fwdarw.2000 nM; over a>12 month
period). The cisplatin-resistant cells, labeled as KB-CR2000 cells,
were found to be tumorigenic, and they retained their FR expression
status in vivo. KB-CR tumors were confirmed to be resistant to
cisplatin therapy since treatment with a high, toxic dose (average
weight loss of 10.3%), as shown in FIG. 3B, of cisplatin produced
no PRs, as shown in FIG. 3A. In contrast, EC1456 was found to be
very active against KB-CR tumors, where 4/5 cures and 1/5 complete
responses were observed. Furthermore, unlike cisplatin, EC1456 did
not cause any weight loss in this cohort of mice.
[0311] Method.
[0312] The therapeutic performance of unconjugated tubulysin B and
TubBH drugs was evaluated against the human KB tumor model, and
anti-tumor as well as body weight changes were compared to that
produced by EC1456. EC1456 produced dose responsive anti-tumor
activity against this model. Complete responses were observed under
treatment conditions that produced little to no weight loss. In
contrast, both unconjugated tubulysin-based drugs failed to yield
any anti-tumor response, even when very toxic doses were
administered to the mice.
TABLE-US-00007 Toxicity Avg. Test Dose level Dose PR CR Cures
Deaths Weight Article (.mu.mol/kg) Schedule (%) (%) (%) (%) Loss
EC1456 0.5 TIW, 3 weeks 60 0 0 0 <5%* 0.67 TIW, 2 weeks 60 20 0
0 <2% 1.0 TIW, 2 weeks 40 0 60 0 <1.5% 2.0 TIW, 2 weeks 0 0
100 0 <3% Tubulysin B 0.1 (4 doses) TIW, 2 weeks 0 0 0 100
>20% 0.2 (3 doses) TIW, 2 weeks 0 0 0 100 >18% 0.5 (1 dose)
TIW, 2 weeks 0 0 0 100 >15% TubBH 0.5 TIW, 2 weeks 0 0 0 0
<5.5% 0.75 TIW, 2 weeks 0 0 0 20 >10% 1.0 (2 doses).sup.1
TIW, 2 weeks 0 0 0 20 >15% *Untreated control group had an
average weight loss of 2.4% .sup.1Group received only 2 doses due
to toxicity.
[0313] These results confirm that despite tubulysin B and TubBH
being highly cytotoxic to cells in culture (typical
IC.sub.50.about.1 nM), both agents yielded dose-limiting toxicities
in mice at levels that did not produce measurable anti-tumor
effect. In contrast, the folate-targeted form of TubBH (EC1456)
produced anti-tumor responses without significant toxicity to mice
bearing well-established human tumor xenografts.
[0314] Method.
[0315] EC1663 is efficacious against against KB tumors in nu/nu
mice and shows 4/4 partial responses in test animals when
administered intravenously at 0.5 .mu.mol/kg TIW, 2 wk compared to
untreated (PBS) controls, as shown in FIG. 4A. EC1663 does not show
any evidence of whole animal toxicity compared to PBS control, as
shown in FIG. 4B.
[0316] Method.
[0317] Maximum tolerated dose (MTD). Compounds described herein
show high MTDs, which are improved over compounds that do not have
linkers comprising one or more unnatural amino acids. For Example,
EC1456 has a MTD of at least 0.51 .mu.mol/kg and EC0531 has a MTD
of 0.33 .mu.mol/kg, a 65% improvement when administered by i.v.,
BIW, 2 wks in female Sprague-Dawley rats. Histopathologic changes
were not observed with doses of EC1456 at or below the MTD.
[0318] Method.
[0319] Folate receptor expressing cells. Compounds described herein
show high activity for folate receptor expressing cells. Compounds
described herein do not show significant binding to folate receptor
negative cells. EC1456 was evaluated from 0.1-100 nM.
TABLE-US-00008 Activity of EC1456 in Various FR+ and FR- Cell Lines
FR EC1456 Competable Cell Line Origin Status Activity* (IC.sub.50)
up to 100 nM KB Human cervical carcinoma +++ 2.3 nM Yes
NCI/ADR-RES-Cl.sub.2 Human ovarian carcinoma ++ 1.4 nM Yes IGROV1
Human ovarian adenocarcinoma + 0.72 nM Yes MDA-MB-231 Human breast
adenocarcinoma + 0.47 nM Yes (triple negative) A549 Human lung
carcinoma - Inactive H23 Human lung adenocarcinoma - Inactive HepG2
Human hepatocellular carcinoma - Inactive AN3CA Human endometrial -
Inactive adenocarcinoma LNCaP Human prostate adenocarcinoma - ~850
nM *EC1456 activity was evaluated from 0.1-100 nM
[0320] Method.
[0321] Compounds described herein exhibit potent in vitro activity
against pathogenic cells, such as KB cells. Compounds described
herein exhibit greater specificity for the folate receptor compared
to compounds that do not include at least one unnatural amino acid.
For Example, EC1456 exhibits ca. 1000-fold specificity for the
folate receptor as determined by folic acid competition
(Specificity=difference in IC.sub.50 between competed group and
non-competed group), and a 4-fold improvement in specificity
compared to EC0531, which does not include a linker L having an
unnatural amino acid.
[0322] Method.
[0323] Compounds described herein exhibit high folate receptor
affinity compared to folic acid (relative affinity=1) in 10%
serum/FDRPMI, potent in vitro activity, potent in vivo activity,
specificity for the folate receptor, and a sufficiently high
therapeutic index over unconjugated tubulysin
TABLE-US-00009 Therapeutic In vitro 50% In vitro index over
Relative IC50 competition specificity In vivo parent Example
Affinity (nM) (nM) (fold) activity tubulysin EC1299 0.29 0.9 700
778 Complete Yes response EC1393 0.25 2.2 600 300 Not Not tested
tested EC1456 0.27 1.5 1416 944 Complete Yes response EC1548 0.23
4.4 350 78 Complete Yes response EC1549 0.90 4.5 350 78 Complete
Yes response EC1586 0.56 Not Not tested Not tested Not Not tested
tested tested EC0531 Not tested 1.5 355 237 Complete Yes
(comparator response example)
[0324] Method.
[0325] Compounds described herein are more stable in plasma than
compounds that do not have a linker comprising at least one
unnatural amino acid. EC1496 releases ca. 50% less drug than the
comparative example EC0746 after a AC dose in rats.
* * * * *